WT Plants Research Articles

The Arabidopsis chromatin regulator MOM1 is a negative component of the defense priming induced by AZA, BABA and PIP.

In plants, the establishment of broad and long-lasting immunity is based on programs that control systemic resistance and immunological memory or "priming". Despite not showing activated defenses, a primed plant induces a more efficient response to recurrent infections. Priming might involve chromatin modifications that allow a faster/stronger activation of defense genes. The Arabidopsis chromatin regulator "Morpheus Molecule 1" (MOM1) has been recently suggested as a priming factor affecting the expression of immune receptor genes. Here, we show that mom1 mutants exacerbate the root growth inhibition response triggered by the key defense priming inducers azelaic acid (AZA), β-aminobutyric acid (BABA) and pipecolic acid (PIP). Conversely, mom1 mutants complemented with a minimal version of MOM1 (miniMOM1 plants) are insensitive. Moreover, miniMOM1 is unable to induce systemic resistance against Pseudomonas sp. in response to these inducers. Importantly, AZA, BABA and PIP treatments reduce the MOM1 expression, but not miniMOM1 transcript levels, in systemic tissues. Consistently, several MOM1-regulated immune receptor genes are upregulated during the activation of systemic resistance in WT plants, while this effect is not observed in miniMOM1. Taken together, our results position MOM1 as a chromatin factor that negatively regulates the defense priming induced by AZA, BABA and PIP.

Functional assessment of AtPAP17; encoding a purple acid phosphatase involved in phosphate metabolism in Arabidopsis thaliana

Purple acid phosphatases (PAPs) includ the largest classes of non-specific plant acid phosphatases. Most characterized PAPs were found to play physiological functions in phosphorus metabolism. In this study, we investigated the function of AtPAP17 gene encoding an important purple acid phosphatase in Arabidopsis thaliana. The full-length cDNA sequence of AtPAP17 gene under the control of CaMV-35S promoter was transferred to the A. thaliana WT plant. The generated homozygote AtPAP17-overexpressed plants were compared by the types of analyses with corresponding homozygote atpap17-mutant plant and WT in both + P (1.2mM) and -P (0mM) conditions. In the + P condition, the highest and the lowest amount of Pi was observed in AtPAP17-overexpressed plants and atpap17-mutant plants by 111% increase and 38% decrease compared with the WT plants, respectively. Furthermore, under the same condition, APase activity of AtPAP17-overexpressed plants increased by 24% compared to the WT. Inversely, atpap17-mutant plant represented a 71% fall compared to WT plants. The comparison of fresh weight and dry weight in the studied plants showed that the highest and the lowest amount of absorbed water belonged to OE plants (with 38 and 12mg plant-1) and Mu plants (with 22 and 7mg plant-1) in + P and -P conditions, respectively. The lack of AtPAP17 gene in the A. thaliana genome led to a remarkable reduction in the development of root biomass. Thus, AtPAP17 could have an important role in the root but not shoot developmental and structural programming. Consequently, this function enables them to absorb more water and eventually associated with more phosphate absorption.

The Proteome and Phosphoproteome Uncovers Candidate Proteins Associated With Vacuolar Phosphate Signal Multipled by Vacuolar Phosphate Transporter 1 (VPT1) in Arabidopsis

Plant vacuoles serve as the primary intracellular compartments for inorganic phosphate (Pi) storage. Passage of Pi across vacuolar membranes plays a critical role in buffering the cytoplasmic Pi level against fluctuations of external Pi and metabolic activities. To gain new insights into the proteins and processes, vacuolar Pi level regulated by vacuolar phosphate transporter 1 (VPT1) in Arabidopsis, we carried out tandem mass tag labeling proteome and phosphoproteome profiling of Arabidopsis WT and vpt1 loss-of-function mutant plants. The vpt1 mutant had a marked reduced vacuolar Pi level and a slight increased cytosol Pi level. The mutant was stunted as reflected in the reduction of the fresh weight compared with WT plants and bolting earlier under normal growth conditions in soil. Over 5566 proteins and 7965 phosphopeptides were quantified. About 146 and 83 proteins were significantly changed at protein abundance or site-specific phosphorylation levels, but only six proteins were shared between them. Functional enrichment analysis revealed that the changes of Pi states in vpt1 are associated with photosynthesis, translation, RNA splicing, and defense response, consistent with similar studies in Arabidopsis. Except for PAP26, EIN2, and KIN10, which were reported to be associated with phosphate starvation signal, we also found that many differential proteins involved in abscisic acid signaling, such as CARK1, SnRK1, and AREB3, were significantly changed in vpt1. Our study illuminates several new aspects of the phosphate response and identifies important targets for further investigation and potential crop improvement.

Down-Regulation of Strigolactone Biosynthesis Gene D17 Alters the VOC Content and Increases Sogatella furcifera Infectivity in Rice

DWARF17 (D17/HTD1) is a well-defined rice strigolactone (SL) biosynthesis gene that influences rice tiller development and the production of rice. To investigate whether SLs play a role in the regulation of rice’s defense against the white-backed planthopper (WBPH, Sogatella furcifera), we compared a SL-biosynthetic defective mutant (d17) with WT rice plants. Our olfactory bioassay results revealed that WBPHs are attracted to d17 plants, which may be attributed to changes in rice volatile substances. Hexanal, a volatile substance, was significantly reduced in the d17 plants, and it was demonstrated that it repelled WBPHs at a concentration of 100 μL/L. Compared to the WT plants, WBPH female adults preferred to oviposit on d17 plants, where the egg hatching rate was higher. The transcript level analysis of defense-associated genes in the JA and SA pathways showed that the expression of OsJAmyb, OsJAZ8, OsPR1a and OsWRKY62 were significantly reduced in d17 plants compared to WT plants following WBPH infection. These findings suggest that silencing the strigolactone biosynthesis gene D17 weakens defenses against S. furcifera in rice.

SUMO protease GmOTSa positively regulates drought tolerance in transgenic tobacco and soybean hairy roots

SUMO protease is important for regulating the balance of SUMOylation and deSUMOylation. Despite the investigations of several SUMO protease genes in model plants, to date, the biological functions of these regulatory enzymes in soybean have yet to be ascertained. In the present study, a SUMO protease GmOTSa was identified in soybean, and was localized in the nucleus with the bona fide SUMO protease activity both in vitro and in vivo. Further RTqPCR analysis showed that GmOTSa was expressed in various tissues and was highly induced by PEG treatment. We demonstrated that GmOTSa conferred drought tolerance in tobacco plants by increasing the germination rate, the fresh and dry weights upon water deficit, and improving the survival rate after rewatering. Overexpression of GmOTSa gene in tobacco lines and soybean hairy roots enhanced drought tolerance by positively regulating ROS scavenging, thus maintained lower ROS content. Moreover, less MDA content and higher total chlorophyll content as well as promoted closure of stomata were detected in the OE plants than in the WT plants in response to drought stress. Conversely, soybean hairy roots with simultaneous knockout of GmOTSa and the homologous gene GmOTSb using the CRISPRCas9 method exhibited reduced drought tolerance. Correspondingly, the expression of drought-related genes showed greater induction upon drought stress in the GmOTSa-overexpressed lines. Overall, these results indicated that GmOTSa may act as a positive regulator of drought responses and therefore could be considered a potential gene for the genetic improvement of drought tolerance.

OsLPXC negatively regulates tolerance to cold stress via modulating oxidative stress, antioxidant defense and JA accumulation in rice.

Exposure of crops to low temperature (LT) during emerging and reproductive stages influences their growth and development. In this study, we have isolated a cold induced, nucleus-localized lipid A gene from rice named OsLPXC, which encodes a protein of 321amino acids. Knockout of OsLPXC resulted in enhance sensitivity to LT stress in rice, with increased accumulation of reactive oxygen species (ROS), malondialdehyde and electrolyte leakage, while expression and activities of antioxidant enzymes were significantly suppressed. The accumulation of chlorophyll content and net photosynthetic rate of knockout plants were also decreased compared with WT under LT stress. The functional analysis of differentially expressed genes (DEGs), showed that numerous genes associated with antioxidant defense, photosynthesis, cold signaling were solely expressed and downregulated in oslpxc plants compared with WT under LT. The accumulation of methyl jasmonate (MeJA) in leave and several DEGs related to the jasmonate biosynthesis pathway were significantly downregulated in OsLPXC knockout plants, which showed differential levels of MeJA regulation in WT and knockout plants in response to cold stress. These results indicated that OsLPXC positively regulates cold tolerance in rice via stabilizing the expression and activities of ROS scavenging enzymes, photosynthetic apparatus, cold signaling genes, and jasmonate biosynthesis.

PIP2;10 Enhances Drought Tolerance via Promoting Water-Retaining Capacity in Populus

Drought is an adverse environmental factor for plant growth and development. Aquaporins play an influential role in water uptake and transport in plants. However, the function of PagPIP2;10 in response to drought stress remains largely unclear. Here, we report that the plasma membrane intrinsic protein PagPIP2;10 was in the cell membrane and induced by dehydration in the poplar 84K hybrids. The overexpression of PagPIP2;10 in poplars enhanced drought tolerance. The PagPIP2;10ox lines maintained a higher water retention content, photosynthetic rate, and proline content. Meanwhile, a lower content of MDA and transpiration and stomatal conductance were observed under drought stress than in that of the WT plants. A further analysis found that the PagPIP2;10ox lines decreased the stomatal aperture and accumulated more ROS in guard cells compared with WT after ABA treatment with the exception that the root hydraulic conductance of the PagPIP2;10ox lines was higher than that of the WT plants. These results imply that PagPIP2;10 played a positive role in enhancing drought stress via enhancing water-retaining capacity under drought stress.

Heterologous overexpression of Apocynum venetum flavonoids synthetase genes improves Arabidopsis thaliana salt tolerance by activating the IAA and JA biosynthesis pathways.

Salt stress is a serious abiotic stress that primarily inhibits plant growth, resulting in severe yield losses. Our previous research found that flavonoids play important roles in A. venetum salt stress tolerance. In response to salt stress, we noted that the flavonoid content was depleted in A. venetum. However, the detailed mechanism is still not clear. In this study, the expression patterns of three flavonoids synthetase genes, AvF3H, AvF3'H, and AvFLS were systemically analyzed under salt stress in A. venetum seedlings. The salt tolerance of transgenic Arabidopsis plants was improved by heterologous overexpression of these synthetase genes. The NBT and DAB staining results as well as H2O2 and O2•- content analysis revealed that under salt stress, ROS molecules were reduced in transgenic plants compared to WT plants, which corresponded to the activation of the antioxidant enzyme system and an increase in total flavonoid content, particularly rutin, eriodictyol, and naringerin in transgenic plants. External application of flavonoids reduced ROS damage in WT plants just like what we observed in the transgenic plants (without the external application). Additionally, our transcriptome analysis demonstrated that auxin and jasmonic acid biosynthesis genes, as well as signaling transduction genes, were primarily activated in transgenic plants under salt stress, leading to activation of the cell wall biosynthesis or modification genes that promote plant growth. As a result, we investigated the mechanism through flavonoids enhance the salt tolerance, offering a theoretical foundation for enhancing salt tolerance in plants.

An R2R3 MYB gene GhMYB3 functions in drought stress by negatively regulating stomata movement and ROS accumulation

MYB transcription factors are one of the largest TF families involved in plant growth and development as well as biotic and abiotic stresses. In this study, we report the identification and functional characterization of a stress-responsive MYB gene (GhMYB3) from drought stress related transcriptome of upland cotton. GhMYB3, belonging to the R2R3-type, has high sequence similarity with AtMYB3 and was localized in the nucleus. Silence of GhMYB3 enhanced the drought tolerance of cotton seedlings and plants, reduced the water loss rate, and enhanced stomatal closure. In addition, GhMYB3i lines exhibited less ROS accumulation, as well as higher antioxidant enzyme activity and increased content of anthocyanins and proanthocyanidins than WT plants after drought stress. The expression level of flavonoid biosynthesis- and stress-related genes were up-regulated in GhMYB3i lines under drought stress condition. These results demonstrated that GhMYB3 acted as a negative regulator in upland cotton response to drought stress by regulating stomatal closure and ROS accumulation.

Yellowhorn Xso-miR5149-XsGTL1 enhances water-use efficiency and drought tolerance by regulating leaf morphology and stomatal density

Yellowhorn (Xanthoceras sorbifolium) is a unique edible woody oil tree species in China. Drought stress is the major yield-limiting factor of yellowhorn. MicroRNAs play an important role in regulating the response of woody plants to drought stress. However, the regulatory function of miRNAs in yellowhorn remains unclear. Here, we first constructed coregulatory networks integrated with miRNAs and their target genes. According to GO function and expression pattern analysis, we selected the Xso-miR5149-XsGTL1 module for further study. Xso-miR5149 is a key regulator of leaf morphology and stomatal density by directly mediating the expression of the transcription factor XsGTL1. Downregulation of XsGTL1 in yellowhorn led to increased leaf area and reduced stomatal density. RNA-seq analysis indicated that downregulation of XsGTL1 increased the expression of genes involved in the negative control of stomatal density, leaf morphology, and drought tolerance. After drought stress treatments, the XsGTL1-RNAi yellowhorn plants were less damaged and had higher water-use efficiency than the WT plants, while destruction of Xso-miR5149 or overexpression of XsGTL1 had the opposite effect. Our findings indicated that the Xso-miR5149-XsGTL1 regulatory module plays a critical role in controlling leaf morphology and stomatal density; hence, it's a potential candidate module for engineering enhanced drought tolerance in yellowhorn.

Wound-induced triacylglycerol biosynthesis is jasmonoy-l-isoleucin and abscisic acid independent.

Triacylglycerol (TAG) plays a significant role during plant stress - it maintains lipid homeostasis. Upon wounding plants accumulate TAG, likely as a storage form of fatty acids (FAs) that originate from damaged membranes. This study asked if this process depends on the two phytohormones jasmonoyl-isoleucine (JA-Ile) and abscisic acid (ABA), which are involved in wound signalling. To analyse regulation of wound-induced TAG accumulation, we used mutants deficient in JA-Ile, with reduced ABA and the myb96 mutant, which is deficient in an ABA-dependent transcription factor. The expression of genes involved in TAG biosynthesis, and TAG content after wounding were analysed via LC-MS and GC-FID, plastidial lipid content in all mentioned mutant lines was also determined. The localization of newly synthesized TAG was investigated using lipid droplet staining. TAG accumulation upon wounding was confirmed as well as the fact that the newly synthesized TAG are mostly composed of polyunsaturated fatty acids. Nevertheless, all tested mutant lines were able to accumulate TAG similar to the WT. We observed differences in reduction of plastidial lipids - in WT plants this was higher than in mutant lines. Newly synthesized TAGs were stored in lipid droplets at and around the wounded area. Our results show that TAG accumulation upon wounding is not dependent on JA-Ile or ABA. The newly synthesized TAG species are composed of unsaturated fatty acids of membrane origin, and most likely serves as a transient energy store.

Identification of Camellia oleifera WRKY transcription factor genes and functional characterization of CoWRKY78.

Camellia oleifera Abel is a highly valued woody edible oil tree, which is endemic to China. It has great economic value because C. oleifera seed oil contains a highproportionof polyunsaturated fatty acids. C. oleifera anthracnose caused by Colletotrichum fructicola, poses a serious threat to C. oleifera growthandyield and causes the benefit of the C. oleifera industry to suffer directly. The WRKY transcription factor family members have been widely characterized as vital regulators in plant responsetopathogeninfection. Until now, the number, type and biological function of C. oleifera WRKY genes are remains unknown. Here, we identified 90C. oleifera WRKY members, which were distributed across 15 chromosomes. C. oleifera WRKY gene expansion was mainly attributedto segmental duplication. We performed transcriptomic analyses to verify the expression patterns of CoWRKYs between anthracnose-resistant and -susceptible cultivars of C. oleifera. These results demonstrated that multiple candidate CoWRKYs can be induced by anthracnose and provide useful clues for their functional studies. CoWRKY78, an anthracnose-induced WRKY gene, was isolated from C. oleifera. It was significantly down-regulated in anthracnose-resistant cultivars. Overexpression of CoWRKY78 in tobacco markedly reduced resistancetoanthracnose than WT plants, as evidenced by more cell death, higher malonaldehyde content and reactive oxygen species (ROS), but lower activities of superoxide dismutase (SOD), peroxidase (POD), as well as phenylalanine ammonia-lyase (PAL). Furthermore, the expression of multiple stress-related genes, which are associated with ROS-homeostasis (NtSOD and NtPOD), pathogen challenge (NtPAL), and pathogendefense (NtPR1, NtNPR1, and NtPDF1.2) were altered in the CoWRKY78-overexpressing plants. These findings increase our understanding of the CoWRKY genes and lay the foundation for the exploration of anthracnose resistance mechanisms and expedite the breeding ofanthracnose-resistant C. oleifera cultivars.

Overexpression of GmWRKY172 enhances cadmium tolerance in plants and reduces cadmium accumulation in soybean seeds.

Cadmium (Cd) stress is a significant threat to soybean production, and enhancing Cd tolerance in soybean is the focus of this study. The WRKY transcription factor family is associated with abiotic stress response processes. In this study, we aimed to identify a Cd-responsive WRKY transcription factor GmWRKY172 from soybean and investigate its potential for enhancing Cd tolerance in soybean. The characterization of GmWRKY172 involved analyzing its expression pattern, subcellular localization, and transcriptional activity. To assess the impact of GmWRKY172, transgenic Arabidopsis and soybean plants were generated and examined for their tolerance to Cd and Cd content in shoots. Additionally, transgenic soybean plants were evaluated for Cd translocation and various physiological stress indicators. RNA sequencing was performed to identify the potential biological pathways regulated by GmWRKY172. GmWRKY172 was significantly upregulated by Cd stress, highly expressed in leaves and flowers, and localized to the nucleus with transcriptional activity. Transgenic plants overexpressing GmWRKY172 showed enhanced Cd tolerance and reduced Cd content in shoots compared to WT. Lower Cd translocation from roots to shoots and seeds was also observed in transgenic soybean. Under Cd stress, transgenic soybean accumulated less malondialdehyde (MDA) and hydrogen peroxide (H2O2) than WT plants, with higher flavonoid and lignin contents, and peroxidase (POD) activity. RNA sequencing analysis revealed that many stress-related pathways were regulated by GmWRKY172 in transgenic soybean, including flavonoid biosynthesis, cell wall synthesis, and peroxidase activity. Our findings demonstrated that GmWRKY172 enhances Cd tolerance and reduces seed Cd accumulation in soybean by regulating multiple stress-related pathways, and could be a promising candidate for breeding Cd-tolerant and low Cd soybean varieties.

GIGANTEA supresses wilt disease resistance by down-regulating the jasmonate signaling in Arabidopsis thaliana.

GIGANTEA (GI) is a plant-specific nuclear protein that plays a pleiotropic role in the growth and development of plants. GI's involvement in circadian clock function, flowering time regulation, and various types of abiotic stress tolerance has been well documented in recent years. Here, the role of GI in response to Fusarium oxysporum (F. oxysporum) infection is investigated at the molecular level comparing Col-0 WT with the gi-100 mutant in Arabidopsis thaliana. Disease progression, photosynthetic parameters, and comparative anatomy confirmed that the spread and damage caused by pathogen infection were less severe in gi-100 than in Col-0 WT plants. F. oxysporum infection induces a remarkable accumulation of GI protein. Our report showed that it is not involved in flowering time regulation during F. oxysporum infection. Estimation of defense hormone after infection showed that jasmonic acid (JA) level is higher and salicylic acid (SA) level is lower in gi-100 compared to Col-0 WT. Here, we show that the relative transcript expression of CORONATINE INSENSITIVE1 (COI1) and PLANT DEFENSIN1.2 (PDF1.2) as a marker of the JA pathway is significantly higher while ISOCHORISMATE SYNTHASE1 (ICS1) and NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1), the markers of the SA pathway, are downregulated in the gi-100 mutants compared to Col-0 plants. The present study convincingly suggests that the GI module promotes susceptibility to F. oxysporum infection by inducing the SA pathway and inhibiting JA signaling in A. thaliana.

Linker histone H1 modulates defense priming and immunity in plants.

Linker H1 histones play an important role in animal and human pathogenesis, but their function in plant immunity is poorly understood. Here, we analyzed mutants of the three canonical variants of Arabidopsis H1 histones, namely H1.1, H1.2 and H1.3. We observed that double h1.1h1.2 and triple h1.1h1.2h1.3 (3h1) mutants were resistant to Pseudomonas syringae and Botrytis cinerea infections. Transcriptome analysis of 3h1 mutant plants showed H1s play a key role in regulating the expression of early and late defense genes upon pathogen challenge. Moreover, 3h1 mutant plants showed enhanced production of reactive oxygen species and activation of mitogen activated protein kinases upon pathogen-associated molecular pattern (PAMP) treatment. However, 3h1 mutant plants were insensitive to priming with flg22, a well-known bacterial PAMP which induces enhanced resistance in WT plants. The defective defense response in 3h1 upon priming was correlated with altered DNA methylation and reduced global H3K56ac levels. Our data place H1 as a molecular gatekeeper in governing dynamic changes in the chromatin landscape of defense genes during plant pathogen interaction.

Brassica rapa orphan gene BR1 delays flowering time in Arabidopsis.

Orphan genes are essential to the emergence of species-specific traits and the process of evolution, lacking sequence similarity to any other identified genes. As they lack recognizable domains or functional motifs, however, efforts to characterize these orphan genes are often difficult. Flowering is a key trait in Brassica rapa, as premature bolting can have a pronounced adverse impact on plant quality and yield. Bolting resistance-related orphan genes, however, have yet to be characterized. In this study, an orphan gene designated BOLTING RESISTANCE 1 (BR1) was identified and found through gene structural variation analyses to be more highly conserved in Chinese cabbage than in other available accessions. The expression of BR1 was increased in bolting resistant Chinese cabbage and decreased in bolting non-resistant type, and the expression of some mark genes were consist with bolting resistance phenotype. BR1 is primarily expressed in leaves at the vegetative growth stage, and the highest BR1 expression levels during the flowering stage were observed in the flower buds and silique as compared to other tissue types. The overexpression of BR1 in Arabidopsis was associated with enhanced bolting resistance under long day (LD) conditions, with these transgenic plants exhibiting significant decreases in stem height, rosette radius, and chlorophyll content. Transcriptomic sequencing of WT and BR1OE plants showed the association of BR1 with other bolting resistance genes. Transcriptomic sequencing and qPCR revealed that six flowering integrator genes and one chlorophyll biosynthesis-related gene were downregulated following BR1 overexpression. Six key genes in photoperiodic flowering pathway exhibited downward expression trends in BR1OE plants, while the expression of floral repressor AtFLC gene was upregulated. The transcripts of these key genes were consistent with observed phenotypes in BR1OE plants, and the results indicated that BR1 may function through vernalization and photoperiodic pathway. Instead, the protein encoded by BR1 gene was subsequently found to localize to the nucleus. Taken together, we first propose that orphan gene BR1 functions as a novel regulator of flowering time, and these results suggested that BR1 may represent a promising candidate gene to support the selective breeding of Chinese cabbage cultivars with enhanced bolting resistance.

Biological insights from multi-omics analysis strategies: Complex pleotropic effects associated with autophagy.

Research strategies that combine molecular data from multiple levels of genome expression (i.e., multi-omics data), often referred to as a systems biology strategy, has been advocated as a route to discovering gene functions. In this study we conducted an evaluation of this strategy by combining lipidomics, metabolite mass-spectral imaging and transcriptomics data from leaves and roots in response to mutations in two AuTophaGy-related (ATG) genes of Arabidopsis. Autophagy is an essential cellular process that degrades and recycles macromolecules and organelles, and this process is blocked in the atg7 and atg9 mutants that were the focus of this study. Specifically, we quantified abundances of ~100 lipids and imaged the cellular locations of ~15 lipid molecular species and the relative abundance of ~26,000 transcripts from leaf and root tissues of WT, atg7 and atg9 mutant plants, grown either in normal (nitrogen-replete) and autophagy-inducing conditions (nitrogen-deficient). The multi-omics data enabled detailed molecular depiction of the effect of each mutation, and a comprehensive physiological model to explain the consequence of these genetic and environmental changes in autophagy is greatly facilitated by the a priori knowledge of the exact biochemical function of the ATG7 and ATG9 proteins.

Overexpression of the autophagy-related gene SiATG8a from foxtail millet (Setaria italica L.) in transgenic wheat confers tolerance to phosphorus starvation

In plants, autophagy plays an important role in regulating intracellular degradation and amino acid recycling in response to nutrient starvation, senescence, and other environmental stresses. Foxtail millet (Setaria italica) shows strong resistance to various abiotic stresses; however, current understanding of the regulation network of abiotic stress resistance in foxtail millet remains limited. In this study, we aimed to determine the autophagy-related gene SiATG8a in foxtail millet. We found that SiATG8a was mainly expressed in the stem and was induced by low-phosphorus (LP) stress. Overexpression of SiATG8a in wheat (Triticum aestivum) significantly increased the grain yield and spike number per m2 under LP treatment compared to those in the WT varieties S366 and S4056. There was no significant difference in the grain P content between SiATG8a-overexpressing wheat and WT wheat under normal phosphorus (NP) and LP treatments. However, the phosphorus (P) content in the roots, stems, and leaves of transgenic plants was significantly higher than that in WT plants under NP and LP conditions. Furthermore, the expression of P transporter genes, such as TaPHR1, TaPHR3, TaIPS1, and TaPT9, in SiATG8a-transgenic wheat was higher than that in WT under LP. Collectively, overexpression of SiATG8a increases the P content of roots, stems, and leaves of transgenic wheat under LP conditions by modulating the expression of P-related transporter gene, which may result in increased grain yield; thus, SiATG8a is a candidate gene for generating transgenic wheat with improved tolerance to LP stress in the field.

Enhancing alfalfa resistance to Spodoptera herbivory by sequestering microRNA396 expression.

Sequestering microRNA396 by overexpression of MIM396 enhanced alfalfa resistance to Spodoptera litura larvae, which may be due to increased lignin content and enhanced low-molecular weight flavonoids and glucosinolates biosynthesis. Alfalfa (Medicago sativa), the most important leguminous forage crop, suffers from the outbreak of defoliator insects, especially Spodoptera litura, resulting in heavy losses in yield and forage quality. Here, we found that the expression of alfalfa microRNA396 (miR396) precursor genes and mature miR396 was significantly up-regulated in wounding treatment that simulates feeding injury by defoliator insects. To verify the function of miR396 in alfalfa resistance to insect, we generated MIM396 transgenic alfalfa plants with significantly down-regulated miR396 expression by Agrobacterium-mediated genetic transformation. The MIM396 transgenic alfalfa plants exhibited improved resistance to Spodoptera litura larvae with increased lignin content but decreased JA accumulation. Most of the miR396 putative target GRF genes were up-regulated in MIM396 transgenic lines, and responded to the wounding treatment. By RNA sequencing analysis, we found that the differentially expressed genes related to insect resistance between WT and MIM396 transgenic plants mainly clustered in biosynthesis pathways in lignin, flavonoids and glucosinolates. In addition to the phenotype of enhanced insect resistance, MIM396 transgenic plants also displayed reduced biomass yield and forage quality. Our results broaden the function of miR396 in alfalfa and provide genetic resources for studying alfalfa insect resistance.

Effect of the Enhanced Production of Chlorophyll b on the Light Acclimation of Tomato.

Tomato (Solanum lycopersicum Mill.) is one of the widely cultured vegetables under protected cultivation, in which insufficient light is one of the major factors that limit its growth, yield, and quality. Chlorophyll b (Chl b) is exclusively present in the light-harvesting complex (LHC) of photosystems, while its synthesis is strictly regulated in response to light conditions in order to control the antenna size. Chlorophyllide a oxygenase (CAO) is the sole enzyme that converts Chl a to Chl b for Chl b biosynthesis. Previous studies have shown that overexpressing CAO without the regulating domain (A domain) in Arabidopsis overproduced Chl b. However, the growth characteristics of the Chl b overproduced plants under different light environmental conditions are not well studied. Considering tomatoes are light-loving plants and sensitive to low light stress, this study aimed to uncover the growth character of tomatoes with enhanced production of Chl b. The A domain deleted Arabidopsis CAO fused with the FLAG tag (BCF) was overexpressed in tomatoes. The BCF overexpressed plants accumulated a significantly higher Chl b content, resulting in a significantly lower Chl a/b ratio than WT. Additionally, BCF plants possessed a lower maximal photochemical efficiency of photosystem II (Fv/Fm) and anthocyanin content than WT plants. The growth rate of BCF plants was significantly faster than WT plants under low-light (LL) conditions with light intensity at 50-70 µmol photons m-2 s-1, while BCF plants grew slower than WT plants under high-light (HL) conditions. Our results revealed that Chl b overproduced tomato plants could better adapt to LL conditions by absorbing more light for photosynthesis but adapt poorly to excess light conditions by accumulating more ROS and fewer anthocyanins. Enhanced production of Chl b is able to improve the growth rate of tomatoes that are grown under LL conditions, indicating the prospect of employing Chl b overproduced light-loving crops and ornamental plants for protected or indoor cultivation.