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.

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.

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.

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.

[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.

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.

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'.

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.

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.

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.

Dephosphorylation of photosystem II proteins and phosphorylation of CP29 in barley photosynthetic membranes as a response to water stress.

Kinetic studies of protein dephosphorylation in barley thylakoid membranes revealed accelerated dephosphorylation of photosystem II (PSII) proteins, and meanwhile rapidly induced phosphorylation of a light-harvesting complex (LHCII) b4, CP29 under water stress. Inhibition of dephosphorylation aggravates stress damages and hampers photosystem recovery after rewatering. This increased dephosphorylation is catalyzed by both intrinsic and extrinsic membrane protein phosphatase. Water stress did not cause any thylakoid destacking, and the lateral migration from granum membranes to stroma-exposed lamellae was only found to CP29, but not other PSII proteins. Activation of plastid proteases and release of TLP40, an inhibitor of the membrane phosphatases, were also enhanced during water stress. Phosphorylation of CP29 may facilitate disassociation of LHCII from PSII complex, disassembly of the LHCII trimer and its subsequent degradation, while general dephosphorylation of PSII proteins may be involved in repair cycle of PSII proteins and stress-response-signaling.

Putative mutation mechanism and light responses of a protochlorophyllide oxidoreductase-less barley mutant NYB.

NYB (Nanchong Yellow Barley) is a Chl-less barley mutant, which is controlled by a recessive nuclear gene. It is the only protochlorophyllide oxidoreductases (POR)-less barley mutant known in the world. The putative mechanism of the mutation and its Chl synthesis and plastid development are studied here. Neither PORC nor an additional copy of porB could be detected in barley. porB mRNAs are normally expressed and correctly spliced in the mutant. However, the import of PORA, PORB, LHCIIb1 (light harvesting complex II b1) and SSU (small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase) proteins to the plastid was greatly hampered in the mutant. We presume that a common translocon is mutated in NYB. The content of the supramolecular light-harvesting POR complex LHPP (light-harvesting NADPH:protochlorophyllide oxidoreductase:protochlorophyllide) and the density of prolamellar bodies in etioplasts are decreased in the mutant. However, no further oxidative damage could be observed for the de-etiolated mutant seedlings after a dark to light shift. Development of the plastid is arrested (less stacking) in NYB, and the mutant becomes more yellowish in high-light conditions, with dwarfing of seedlings and decreased yield. The physiological significance and developmental roles of POR proteins and LHPP in barley cells are discussed.

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.

Light regulation to chlorophyll synthesis and plastid development of the chlorophyll-less golden-leaf privet.

Ligustrum vicaryi L. is a hybrid of Ligustrum ovalifolium Hassk. var. aureo-marginatum and Ligustrum vulgale L., and displays a chlorophyll-less phenotype. Therefore it is widely used as a horticultural shrub because of its golden-color leaves. Its putative mechanism, light responses, chlorophyll synthesis and plastid development were studied. L. vicaryi has a higher level of 5-aminolevulinic acid (ALA), but lower levels of chlorophylls compared with L. quihoui. The yellowish phenotype of L. vicaryi upper leaves could be attributed to their hampered conversion from chlorophyllide into chlorophyll a. Despite the enhanced ALA level and the decreased thylakoid stacking in plastids, L. vicaryi golden leaves contain normal levels of Lhcb transcripts and photosystem apoproteins. Furthermore, reactive oxygen species (ROS) accumulation is almost the same in L. vicaryi and L. quihoui. The golden leaves often turn green and the contents of chlorophylls increase with decreasing light intensity. Dynamic changes of chlorophyll-synthesis-system under the light transition were also analyzed.

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.

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.

Phosphorylation of photosynthetic antenna protein CP29 and photosystem II structure changes in monocotyledonous plants under environmental stresses.

Kinetic studies of protein dephosphorylation in thylakoid membranes showed that the minor light-harvesting antenna protein CP29 could be phosphorylated in barley (C3) and maize (C4) seedlings, but not in spinach under water [Liu, W. J., et al. (2009) Biochim. Biophys. Acta 1787, 1238-1245], salt, or cold stress [Pursiheimo, S., et al. (2003) Plant Cell Environ. 26, 1995-2003], suggesting that phosphorylation of CP29 is a general phenomenon in monocots, but not in dicots under environmental stresses. Abscisic acid (ABA), reactive oxygen species (ROS), salicylic acid (SA), jasmonic acid (JA), ethylene (ET), NO, and the scavenger of H(2)O(2) had weak effects on CP29 phosphorylation. However, three protein kinase inhibitors, U0126, W7, and K252a (for mitogen-activated protein kinase, Ca(2+)-dependent protein kinase, and Ser/Thr protein kinases, respectively), decrease the level of CP29 phosphorylation in barley apparently under environmental stresses. Therefore, these three protein kinases are involved in CP29 phosphorylation. We also found that most CP29 phosphorylation was accompanied by its lateral migration from granum membranes to stroma-exposed thylakoid regions, and the instability of PSII supercomplexes and LHCII trimers under environmental stresses.

Brassinosteroids counteract abscisic acid in germination and growth of Arabidopsis.

Brassinosteroids (BRs) are involved in multiple plant growth and development processes, such as cell elongation, photomorphogenesis, flowering time control, and stress responses. The phytohormone abscisic acid (ABA) is crucial to plant development and adaptation to stressful environments. The receptors and pathways of BRs and ABA have been deeply studied. But the relationship between them remained largely unknown and there are only few reports about it. Our experiments showed that the BR-deficient and BR-insensitive Arabidopsis mutants det2, bri1-5 and bri1-9 were more sensitive to ABA than the wild type (Ws-2), especially the det2 and bri1-9 mutants. Germination, hypocotyl and root elongation, and stomatal apertures of the mutants were more severely inhibited by ABA. All the results suggest that BRs counteract ABA in regulating plant growth, and the interaction may be complicated. The possible mechanisms are discussed.

Lack of salicylic acid in Arabidopsis protects plants against moderate salt stress.

Previous studies showed that salicylic acid (SA)-deficient transgenic Arabidopsis expressing the salicylate hydroxylase gene NahG had a higher tolerance to moderate salt stress. SA may potentiate the stress response of germination and growth of Arabidopsis seedlings by inducing reactive oxygen species (ROS). However, the detailed mechanism for a better adaption of NahG plants to moderate salt stress is largely unknown. In the present study we found that a higher GSH/GSSG (glutathione/oxidized glutathione) ratio and ASA/DHA (ascorbic acid/dehydroascorbate) ratio in NahG plants during the stress may be the key reason for their stress-tolerance advantage. NahG plants actually could not produce more active antioxidant enzymes than the wild-type ones under natural conditions, but maintain higher activities of glutathione reductase (GR) and dehydroascorbate reductase (DHAR) during the stress. Hereby, the reduced glutathione and reduced ascorbic acid contents are higher in NahG plants under salt stress. However, NahG plants do not adapt better under severe salt stress. All antioxidant enzyme activities, GSH/GSSG ratio and ASA/DHA ratio declined substantively at 400 mM NaCl stress in both NahG and wild-type seedlings.

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.