Singlet oxygen induces cell wall thickening and stomatal density reducing by transcriptome reprogramming.

Singlet oxygen (1O2) has a very short half-life of 10-5 s; however, it is a strong oxidant that causes growth arrest and necrotic lesions on plants. Its signaling pathway remains largely unknown. The Arabidopsis flu (fluorescent) mutant accumulates a high level of 1O2 and shows drastic changes in nuclear gene expression. Only two plastid proteins, EX1 (executer 1) and EX2 (executer 2), have been identified in the singlet oxygen signaling. Here, we found that the transcription factor abscisic acid insensitive 4 (ABI4) binds the promoters of genes responsive to 1O2-signals. Inactivation of the ABI4 protein in the flu/abi4 double mutant was sufficient to compromise the changes of almost all 1O2-responsive-genes and rescued the lethal phenotype of flu grown under light/dark cycles, similar to the flu/ex1/ex2 triple mutant. In addition to cell death, we reported for the first time that 1O2 also induces cell wall thickening and stomatal development defect. Contrastingly, no apparent growth arrest was observed for the flu mutant under normal light/dim light cycles, but the cell wall thickening (doubled) and stomatal density reduction (by two-thirds) still occurred. These results offer a new idea for breeding stress tolerant plants.

Soybean balanced the growth and defense in response to SMV infection under different light intensities.

Light is essential for the growth and defense of soybean. It is not clear how soybeans adjust their defenses to different light environments with different cropping patterns. The mechanism of soybean response to Soybean mosaic virus (SMV) infection under different light intensities was analyzed by RNA-seq sequencing method. Enrichment analysis illustrated that most defense-related genes were down-regulated in the dark and the shade, and up-regulated under hard light and normal light. Soybean can resist SMV infection mainly by activating salicylic acid signaling pathway. Light is essential for activating salicylic acid defense signaling pathways. With the increase of light intensity, the oxidative damage of soybean leaves was aggravated, which promoted the infection of virus. When light was insufficient, the growth of soybean was weak, and the plant-pathogen interaction pathway, MAPK pathway and hormone defense pathway in infected soybean was inhibited. Under hard light, some defense genes in infected soybean were down-regulated to reduce the degree of oxidative damage. The expression of differentially expressed genes was verified by real-time fluorescence quantitative RT-PCR. In order to adapt to the change of light intensity, soybean balanced allocation of resources between growth and defense through a series regulation of gene expression. The results of this study will provide a theoretical basis for the research of SMV resistance in intercropping soybean.

Nitrogen allocation for CO2 fixation promotes nitrogen use in the photosynthetic compensation of soybean under heterogeneous light after a pre-shading.

The spatial and temporal distribution of sunlight around plants is constantly changing in natural and farmland environments. Previous studies showed that the photosynthesis of crops responds significantly to heterogeneous light conditions in fields. However, the underlying mechanisms remain unclear. In the present study, soybean plants were treated by heterogeneous light after a pre-shading (SH-HL) to simulate the light condition in relay strip intercropping. Gas exchange and nitrogen (N) of leaves were measured to evaluate the photosynthetic performance, as well as photosynthetic N- and water-use efficiency (PNUE and PWUE). Chlorophylls (Chl) and Rubisco were analyzed as representative photosynthetic N components. Results suggest that SH-HL treated soybean exhibited evident photosynthetic compensation as the net photosynthetic rate (Pn) increased significantly in unshaded leaves, from which the export of photosynthates was enhanced. Under SH-HL, leaf N concentration remained relatively stable in unshaded leaves. While Chl concentration decreased but Rubisco concentration increased in unshaded leaves, indicating preferential allocation of leaf N for CO2 fixation. Results also showed that PNUE increased and PWUE decreased in unshaded leaves under SH-HL. Therefore, we suggest that within-leaf N allocation for CO2 fixation in unshaded leaves rather than within-plant N distribution to unshaded leaves drives the photosynthetic compensation under heterogeneous light after a pre-shading. However, enhanced water loss from unshaded leaves is a cost for efficient N-use under these conditions. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01392-8.

Photosynthetic compensation of maize in heterogeneous light is impaired by restricted photosynthate export.

When a plant is exposed to heterogeneous light, the photosynthesis of unshaded leaves is often stimulated to compensate for the decline in photosynthesis of shaded leaves, i.e., photosynthetic compensation. However, a decline of photosynthesis in unshaded leaves, which means an impairment of photosynthetic compensation, has also been widely reported. Herein, two cultivars of maize (Zea mays L.), 'Rongyu1210' (RY) and 'Zhongdan808' (ZD), were studied comparatively. Both cultivars performed evident photosynthetic compensation under heterogeneous light (HL) as the light phase begins (8:30 a.m.). However, as the light phase continues (10:30 a.m.), an impairment of photosynthetic compensation took place in HL-treated ZD, but not in HL-treated RY. For both cultivars, nitrogen content of unshaded leaves was higher under HL, indicating a preferential nitrogen distribution towards unshaded leaves. This is related to the photosynthetic compensation but not the cause of the impairment. In addition, no obvious difference was found in the response of photosynthates (sucrose and starch) to HL between cultivars at 8:30 a.m. However, at 10:30 a.m., the content of photosynthates decreased significantly in unshaded leaves of HL-treated RY, along with increased abundances of both sucrose transporters (SUTs) and H+-ATPase (EC 7.1.2.1). In contrast, it increased along with decreased abundances of SUTs and H+-ATPase in HL-treated ZD. These results suggest that the photosynthetic compensation is impaired when photosynthates export of unshaded leaves is restricted. This suggestion is further confirmed by the results of 13C labeling and dry weight detection on young leaves as 'sink'.

Potential importance of malate diffusion in the response of maize photosynthesis to heterogeneous light.

It is well known that the photosynthetic performance of a leaf is highly dependent on the systemic regulation from distal parts within a plant under light heterogeneity. However, there are few studies focusing on C4-specific processes. In the present study, two cultivars of maize (Zea mays L.), 'Rongyu 1210' (RY) and 'Zhongdan 808' (ZD), were treated with heterogeneous light (HL). The net photosynthetic rate (Pn) of newly developed leaves was found to increase in HL-treated RY, while it decreased in HL-treated ZD. Result also showed a negative correlation between the Pn and the content of malate, a key metabolite in C4 photosynthesis, in these two cultivars. In HL-treated ZD, malate content increased with a decline in the abundance of NADP-malic enzyme (EC 1.1.1.40), suggesting that less malate was decarboxylated. Moreover, a restriction of malate diffusion is proposed in HL-treated ZD, since the interface length between mesophyll cells (MC) and bundle sheath cells (BSC) decreased. In contrast, malate diffusion and subsequent decarboxylation in HL-treated RY should be stimulated, due to an increase in the abundance of NADP-malate dehydrogenase (EC 1.1.1.82) and a decline in the content of malate. In this case, malate diffusion from MC to BSC should be systemically stimulated, thereby facilitating C4 photosynthesis of a maize leaf in heterogeneous light. While if it is systemically restricted, C4 photosynthesis would be suppressed.

Comparative study on the different responses of maize photosynthesis to systemic regulation under light heterogeneity.

Photosynthetic performance of a leaf is widely recognized to be systemically regulated by distal parts within the same plant. However, the effects of systemic regulation on different plant materials cannot be generalized. In this work, two cultivars of maize (Zea mays L.), 'Rongyu 1210' (RY) and 'Zhongdan 808' (ZD), were selected for a comparative study on the different responses of photosynthesis to light-dependent systemic regulation. After the growth of plants in heterogeneous light, the net photosynthetic rate of newly developed leaves increased in RY but decreased in ZD. A distinct capacity of CO2 fixation and assimilation between these two cultivars is also suggested. In ZD, the area of vascular bundles declined obviously, suggesting a restriction on carbohydrate export, which is also indicated by an increase in starch content. Resulting excessive accumulation of carbohydrates is proposed to inhibit the carbon assimilation, and eventually the photosynthesis. A decline in the area of bundle sheath cells also suggests a restriction on carbon assimilation. In contrast, these restrictions were unlikely to present in RY. This study reveals that the response of leaf photosynthetic performance to light heterogeneity is largely dependent on the systemic regulation of carbon assimilation, as well as carbohydrate export in maize.

Involvement of carbohydrates in long-term light-dependent systemic regulation on photosynthesis of maize under light heterogeneity.

It is widely recognized that different parts of a plant can communicate with each other via light-dependent long-distance signaling under heterogeneous light conditions. However, the mechanism of such systemic signaling has not been revealed yet. Our studies on different species suggest the involvement of carbohydrates in light-dependent systemic regulation between different parts of a plant under both short- and long-term light heterogeneity. Leaves exposed to better light condition perform enhanced photosynthetic capacity, and act to compensate for the decline in photosynthesis of other leaves under bad light condition within the same plant. This kind of compensatory photosynthesis has a close relationship to the distribution of carbohydrates, and can be regarded as an integrative strategy to make efficient use of sunlight at the whole-plant level.

Two-factor ANOVA of SSH and RNA-seq analysis reveal development-associated Pi-starvation genes in oilseed rape.

MAIN CONCLUSION: The 5-leaf-stage rape seedlings were more insensitive to Pi starvation than that of the 3-leaf-stage plants, which may be attributed to the higher expression levels of ethylene signaling and sugar-metabolism genes in more mature seedlings. Traditional suppression subtractive hybridization (SSH) and RNA-Seq usually screen out thousands of differentially expressed genes. However, identification of the most important regulators has not been performed to date. Here, we employed two methods, namely, a two-round SSH and two-factor transcriptome analysis derived from the two-factor ANOVA that is commonly used in the statistics, to identify development-associated inorganic phosphate (Pi) starvation-induced genes in Brassica napus. Several of these genes are related to ethylene signaling (such as EIN3, ACO3, ACS8, ERF1A, and ERF2) or sugar metabolism (such as ACC2, GH3, LHCB1.4, XTH4, and SUS2). Although sucrose and ethylene may counteract each other at the biosynthetic level, they may also work synergistically on Pi-starvation-induced gene expression (such as PT1, PT2, RNS1, ACP5, AT4, and IPS1) and root acid phosphatase activation. Furthermore, three new transcription factors that are responsive to Pi starvation were identified: the zinc-finger MYND domain-containing protein 15 (MYND), a Magonashi family protein (MAGO), and a B-box zinc-finger family salt-tolerance protein. This study indicates that the two methods are highly efficient for functional gene screening in non-model organisms.

Genetic map construction and QTL analysis of leaf-related traits in soybean under monoculture and relay intercropping.

Soybean (Glycine max L.) is an important food and oil crop widely planted by intercropping in southwest China. The shade caused by intercropping changes plant growth traits, such as soybean leaf and dry mass, thereby reducing yields. To improve the yield and elucidate the genetic mechanism of the leaf-related traits in intercropped soybeans, we measured the F6:7-8 recombinant inbred lines (RILs) derived from the cross of 'Nandou 12' and 'Jiuyuehuang' for six leaf-related traits under monoculture and relay intercropping in 2015 and 2016. We found 6366 single-nucleotide polymorphisms (SNPs) markers that covered the whole genome of soybean distributed in 20 linkage groups, which spanned 2818.67 cM with an average interval of 0.44 cM between adjacent markers. Nineteen quantitative trait loci (QTLs) were detected in two environments in 2 years. Three candidate genes associated to leaf-related traits were found according to gene expression and GO enrichment analyses. These results revealed the susceptibility of leaf phenotype to shading and helped elucidate the mechanisms that control leaf-related traits.

Fungal Diversity in Field Mold-Damaged Soybean Fruits and Pathogenicity Identification Based on High-Throughput rDNA Sequencing.

Continuous rain and an abnormally wet climate during harvest can easily lead to soybean plants being damaged by field mold (FM), which can reduce seed yield and quality. However, to date, the underlying pathogen and its resistance mechanism have remained unclear. The objective of the present study was to investigate the fungal diversity of various soybean varieties and to identify and confirm the FM pathogenic fungi. A total of 62,382 fungal ITS1 sequences clustered into 164 operational taxonomic units (OTUs) with 97% sequence similarity; 69 taxa were recovered from the samples by internal transcribed spacer (ITS) region sequencing. The fungal community compositions differed among the tested soybeans, with 42 OTUs being amplified from all varieties. The quadratic relationships between fungal diversity and organ-specific mildew indexes were analyzed, confirming that mildew on soybean pods can mitigate FM damage to the seeds. In addition, four potentially pathogenic fungi were isolated from FM-damaged soybean fruits; morphological and molecular identification confirmed these fungi as Aspergillus flavus, A. niger, Fusarium moniliforme, and Penicillium chrysogenum. Further re-inoculation experiments demonstrated that F. moniliforme is dominant among these FM pathogenic fungi. These results lay the foundation for future studies on mitigating or preventing FM damage to soybean.

Nitric oxide induces monosaccharide accumulation through enzyme S-nitrosylation.

Nitric oxide (NO) is extensively involved in various growth processes and stress responses in plants; however, the regulatory mechanism of NO-modulated cellular sugar metabolism is still largely unknown. Here, we report that NO significantly inhibited monosaccharide catabolism by modulating sugar metabolic enzymes through S-nitrosylation (mainly by oxidizing dihydrolipoamide, a cofactor of pyruvate dehydrogenase). These S-nitrosylation modifications led to a decrease in cellular glycolysis enzymes and ATP synthase activities as well as declines in the content of acetyl coenzyme A, ATP, ADP-glucose and UDP-glucose, which eventually caused polysaccharide-biosynthesis inhibition and monosaccharide accumulation. Plant developmental defects that were caused by high levels of NO included delayed flowering time, retarded root growth and reduced starch granule formation. These phenotypic defects could be mediated by sucrose supplementation, suggesting an essential role of NO-sugar cross-talks in plant growth and development. Our findings suggest that molecular manipulations could be used to improve fruit and vegetable sweetness.

Metabolism variation and better storability of dark- versus light-coloured soybean (Glycine max L. Merr.) seeds.

The effects of storage duration on the seed germination and metabolite profiling of soybean seeds with five different coloured coats were studied. Their germination, constituents and transcript expressions of isoflavones and free fatty acids (FFAs) were compared using chromatographic metabolomic profiling and transcriptome sequencing. The seed water content was characterized using nuclear magnetic resonance (NMR) relaxometry. Results showed that dark-coloured seeds were less inactivated than light-coloured seeds. The aglycone and ß-glucoside concentrations of upstream constituents increased significantly, whereas the acetylglucosides and malonylglucosides of downstream constituents decreased with an increase in the storage period. FFAs increased considerably in the soybean seeds as a result of storage. These results indicate that dark-coloured soybean seeds have better storability than light-coloured seeds, and seed water content plays a role in seed inactivation. It was concluded that there are certain metabolic regularities that are associated with different coloured seed coats of soybeans under storage conditions.

[Relationship between cellulose synthesis metabolism and lodging resistance in intercropping soybean at seedling stage].

Physical characteristics of stem are closely relative to the crop lodging. Increase of stem strength is conducive to resolve the problem of lodging. Three soybean cultivars with different shade tolerance were planted under maize-soybean intercropping and soybean monocropping, respectively. Physiological and biochemical indices including cellulose, soluble sugar, sucrose, starch contents and enzyme activity were investigated to assess the snapping resistance and lodging resistance of the stems of soybean seedling, and snapping- and lodging-resistance indices were calculated for further verification. Furthermore, relationship analyses between these factors and the lodging of inter-cropped soybean showed that the intercropping soybean lodged seriously, the snapping resistance, lodging resistance index, contents of cellulose, soluble sugar, sucrose, starch and activities of the related enzymes were significantly lower than monocropping soybean at seedling stage. The three soybean cultivars showed different phenotypes in intercropping condition. The snapping-resistant Nandou12 with strong shade-tolerant traits was the most lodging-resistant phenotype, and it also harbored high contents of cellulose, soluble sugar, sucrose, starch and active enzymes. The lodging resistance index, cellulose content of the stems of intercropped soybean seedling were significantly positively correlated with the snapping resistance, and were significantly negatively correlated with the actual lodging percentage. The activities of sucrose phosphate synthase (SPS) , sucrose synthase (SS) and neutral invertase (NI) were positively correlated with sucrose is content, but not the acid invertase (AI). The activities of SPS, NI and SS were positively correlated with cellulose content, but not Al. In a word, the high activities of SPS and SS in the soybean stem were the enzymatic basis to maintain relatively higher cellulose and sucrose content, which is conducive to improve the stem-sfrength and enhance the lodging resistance ability in intercropping condition. Effects of different light conditions on cellulose metabolic mechanism of soybean seedling stems, lodging resistant characteristics of soybean seedlings studied in the corn-soybean intercropping system provided a basis for screening more shade-tolerant soybean variety.

[Effect of fertilization levels on soil microorganism amount and soil enzyme activities].

Field experiments were conducted in Shangluo pharmaceutical base in Shaanxi province to study the effect of nitrogen, phosphorus and potassium in different fertilization levels on Platycodon grandiflorum soil microorganism and activities of soil enzyme, using three-factor D-saturation optimal design with random block design. The results showed that N0P2K2, N2P2K0, N3P1K3 and N3P3K1 increased the amount of bacteria in 0-20 cm of soil compared with N0P0K0 by 144.34%, 39.25%, 37.17%, 53.58%, respectively. The amount of bacteria in 2040 cm of soil of N3P1K3 increased by 163.77%, N0P0K3 increased the amount of soil actinomycetes significantly by 192.11%, while other treatments had no significant effect. N2P0K2 and N3P1K3 increased the amounts of fungus significantly in 0-20 cm of soil compared with N0P0K0, increased by 35.27% and 92.21%, respectively. N3P0K0 increased the amounts of fungus significantly in 20-40 cm of soil by 165.35%, while other treatments had no significant effect. All treatments decrease soil catalase activity significantly in 0-20 cm of soil except for N2P0K2, and while N2P2K0 and NPK increased catalase activity significantly in 2040 cm of soil. Fertilization regime increased invertase activity significantly in 2040 cm of soil, and decreased phosphatase activity inordinately in 0-20 cm of soil, while increased phosphatase activity in 2040 cm of soil other than N1P3K3. N3P0K0, N0P0K3, N2P0K2, N2P2K0 and NPK increased soil urease activity significantly in 0-20 cm of soil compared with N0P0K0 by 18.22%, 14.87%,17.84%, 27.88%, 24.54%, respectively. Fertilization regime increased soil urease activity significantly in 2040 cm of soil other than N0P2K2.

[Seedling index of Salvia miltiorrhiza and its simulation model].

Through the correlation analysis on the quantitative traits and their ratios of Salvia miltiorrhiza seedlings and seedling quality, a series of representative indices reflecting the seedling quality of the plant species were determined, and the seedling index suitable to the S. miltiorrhiza seedlings was ascertained by correlation degree analysis. Meanwhile, based on the relationships between the seedling index and the air temperature, solar radiation and air humidity, a simulation model for the seedling index of S. miltiorrhiza was established. The experimental data of different test plots and planting dates were used to validate the model. The results showed that the root diameter, stem diameter, crown dry mass, root dry mass, and plant dry mass had significant positive relationships with the other traits, and could be used as the indicators of the seedling's health. The seedling index of S. miltiorrhiza could be calculated by (stem diameter/root diameter + root dry mass/crown dry mass) x plant dry mass. The stem diameter, root dry mass, crown dry mass and plant dry mass had higher correlations with the seedling index, and thus, the seedling index determined by these indicators could better reflect the seedling's quality. The coefficient of determination (R2) between the predicted and measured values based on 1:1 line was 0.95, and the root mean squared error (RMSE) was 0.15, indicating that the model established in this study could precisely reflect the quantitative relationships between the seedling index of S. miltiorrhiza and the environmental factors.

Red blood cell extrudes nucleus and mitochondria against oxidative stress.

Mammal red blood cells (erythrocytes) contain neither nucleus nor mitochondria. Traditional theory suggests that the presence of a nucleus would prevent big nucleated erythrocytes to squeeze through these small capillaries. However, nucleus is too small to hinder erythrocyte deformation. And, there is no sound reason to abandon mitochondria for the living cells. Here, we found that mammal erythrocyte reactive oxygen species (ROS) levels kept stable under diabetes, ischemia reperfusion, and malaria conditions or in vitro sugar/heme treatments, whereas bird erythrocyte ROS levels increased dramatically in these circumstances. Nuclear and mitochondrial extrusion may help mammal erythrocytes to better adapt to high-sugar and high-heme conditions by limiting ROS generation.

Mammal cells double their total RNAs against diabetes, ischemia reperfusion and malaria-induced oxidative stress.

Total cellular RNA level is stable usually, although it may increase gradually during growth or decrease gradually under certain stressors. However, we found that mammal cell RNAs could be doubled within 24 h in response to free heme accumulation (ischemia reperfusion and malaria infection) or a high level of glucose treatment (diabetes). Clinical investigations in rats showed that pretreatment with heme (24 h for doubling total RNAs) alleviated oxidative damages caused by diabetes, and pretreatment with glucose (24 h for trebling total RNAs) alleviated oxidative damages caused by ischemia reperfusion or malaria infection. Therefore, this rapid RNA amplification may play an important role in mammal adaptation to diabetes, ischemia reperfusion and malaria infection-derived oxidative stress. This rapid RNA amplification is derived from glucose and heme, but not from their accompanying reactive oxygen species. Hexokinases endure glucose-derived reactive oxygen species accumulation but are not related glucose-derived RNA amplification. In contrast, the TATA box-binding protein (TBP) mediates all glucose- and heme-induced RNA amplification in mammal cells.

Plant-like proteins in protozoa, metazoa and fungi imply universal plastid endosymbiosis.

In recent years, plant-like proteins in protozoa, metazoa and fungi have been identified. Analysis of them suggests that for millions of years universal plastid endosymbiosis and gene transfer occurred in ancestors of metazoa/fungi, and some transferred fragments have been reserved till now even in modern mammals. Most eukaryotes once contained plastids in the ancient era, and some of them lost plastids later. Functions of homologues in cyanobacterial genomes and eukaryotic genomes are in consensus, and are most involved in organic compound metabolism. With emergence of organelles and subcellular structures in the eukaryotic cell, the locations of these proteins diversified. Furthermore, some novel functions were adopted, especially in vertebrates. Analysis also implies that plastids acquired through a mechanism of secondary endosymbiosis may be preserved even until the multicellular era in simple animals. Phylogenetic trees of some proteins suggest that in ancient times the common ancestor of photosynthetic protist Euglena and parasite Trypanosoma once engulfed a green alga, and then it lost the plastid, but recently some euglenids engulfed algae again. Plastid endosymbiosis is a more general process than we originally thought, and may happen more than one time in one species.

Effects of light on cyanide-resistant respiration and alternative oxidase function in Arabidopsis seedlings.

Mitochondrial alternative oxidase (AOX), the unique respiratory terminal oxidase in plants, catalyzes the energy wasteful cyanide (CN)-resistant respiration and plays a role in optimizing photosynthesis. Although it has been demonstrated that leaf AOX is upregulated after illumination, the in vivo mechanism of AOX upregulation by light and its physiological significance are still unknown. In this report, red light and blue light-induced AOX (especially AOX1a) expressions were characterized. Phytochromes, phototropins and cryptochromes, all these photoreceptors mediate the light-response of AOX1a gene. When aox1a mutant seedlings were grown under a high-light (HL) condition, photobleaching was more evident in the mutant than the wild-type plants. More reactive oxygen species (ROS) accumulation and inefficient dissipation of chloroplast reducing-equivalents in aox1a mutant may account for its worse adaptation to HL stress. When etiolated seedlings were exposed to illumination for 4 h, chlorophyll accumulation was largely delayed in aox1a plants. We first suggest that more reduction of the photosynthetic electron transport chain and more accumulation of reducing-equivalents in the mutant during de-etiolation might be the main reasons.