Transgenic Seedlings Research Articles

Comparative physiological, metabolomic, and transcriptomic analyses reveal mechanisms of apple dwarfing rootstock root morphogenesis under nitrogen and/or phosphorus deficient conditions.

Nitrogen (N) and phosphorus (P) are essential phytomacronutrients, and deficiencies in these two elements limit growth and yield in apple (Malus domestica Borkh.). The rootstock plays a key role in the nutrient uptake and environmental adaptation of apple. The objective of this study was to investigate the effects of N and/or P deficiency on hydroponically-grown dwarfing rootstock 'M9-T337' seedlings, particularly the roots, by performing an integrated physiological, transcriptomics-, and metabolomics-based analyses. Compared to N and P sufficiency, N and/or P deficiency inhibited aboveground growth, increased the partitioning of total N and total P in roots, enhanced the total number of tips, length, volume, and surface area of roots, and improved the root-to-shoot ratio. P and/or N deficiency inhibited influx into roots, and H+ pumps played a important role in the response to P and/or N deficiency. Conjoint analysis of differentially expressed genes and differentially accumulated metabolites in roots revealed that N and/or P deficiency altered the biosynthesis of cell wall components such as cellulose, hemicellulose, lignin, and pectin. The expression of MdEXPA4 and MdEXLB1, two cell wall expansin genes, were shown to be induced by N and/or P deficiency. Overexpression of MdEXPA4 enhanced root development and improved tolerance to N and/or P deficiency in transgenic Arabidopsis thaliana plants. In addition, overexpression of MdEXLB1 in transgenic Solanum lycopersicum seedlings increased the root surface area and promoted acquisition of N and P, thereby facilitating plant growth and adaptation to N and/or P deficiency. Collectively, these results provided a reference for improving root architecture in dwarfing rootstock and furthering our understanding of integration between N and P signaling pathways.

Genome-wide identification of the AcMADS-box family and functional validation of AcMADS32 involved in carotenoid biosynthesis in Actinidia.

MADS-box is a large transcription factor family in plants and plays a crucial role in various plant developmental processes; however, it has not been systematically analyzed in kiwifruit. In the present study, 74 AcMADS genes were identified in the Red5 kiwifruit genome, including 17 type-I and 57 type-II members according to the conserved domains. The AcMADS genes were randomly distributed across 25 chromosomes and were predicted to be mostly located in the nucleus. A total of 33 fragmental duplications were detected in the AcMADS genes, which might be the main force driving the family expansion. Many hormone-associated cis-acting elements were detected in the promoter region. Expression profile analysis showed that AcMADS members had tissue specificity and different responses to dark, low temperature, drought, and salt stress. Two genes in the AG group, AcMADS32 and AcMADS48, had high expression levels during fruit development, and the role of AcMADS32 was further verified by stable overexpression in kiwifruit seedlings. The content of α-carotene and the ratio of zeaxanthin/β-carotene was increased in transgenic kiwifruit seedlings, and the expression level of AcBCH1/2 was significantly increased, suggesting that AcMADS32 plays an important role in regulating carotenoid accumulation. These results have enriched our understanding of the MADS-box gene family and laid a foundation for further research of the functions of its members during kiwifruit development.

A long non-coding RNA PelncRNA1 is involved in Phyllostachys edulis response to UV-B stress.

Phyllostachys edulis (moso bamboo) is China's most widespread bamboo species, with significant economic and ecological values. Long non-coding RNA (lncRNA) is a type of regulatory RNA that is longer than 200 nucleotides and incapable of encoding proteins, and is frequently involved in regulating biotic and abiotic stress and plant development. However, the biological functions of lncRNA in moso bamboo are unknown. In this study, a lncRNA (named PelncRNA1) differentially expressed following UV-B treatment was discovered in the whole transcriptome sequencing database of moso bamboo. The target genes were filtered and defined by correlation analysis of PelncRNA1 and gene expression pattern. The expression levels of PelncRNA1 and its target genes were verified using qRT-PCR. The results demonstrated that the expression levels of PelncRNA1 and its target genes increased during UV-B treatment. In Arabidopsis transgenic seedlings and moso bamboo protoplasts, PelncRNA1 was discovered to influence the expression of its target genes when overexpressed. In addition, transgenic Arabidopsis showed higher tolerance to UV-B stress. These results suggest that PelncRNA1 and its target genes are involved in the response of moso bamboo to UV-B stress. The novel findings would contribute to our understanding of how lncRNAs regulate the response to abiotic stresses in moso bamboo.

OsMBTB32, a MATH-BTB domain-containing protein that interacts with OsCUL1s to regulate salt tolerance in rice.

MATH-BTB proteins are involved in a variety of cellular processes that regulate cell homeostasis and developmental processes. Previous studies reported the involvement of BTB proteins in the development of various organs in plants; however, the function of BTB proteins in salt stress is less studied. Here, we found a novel MATH-BTB domain-containing OsMBTB32 protein that was highly expressed in leaf, root, and shoot. The up-regulation of the OsMBTB32 transcript in 2-week-old seedlings under salt stress suggests the significant role of the OsMBTB32 gene in salinity. The OsMBTB32 transgenic seedlings (OE and RNAi) exhibited significant differences in various phenotypes, including plumule, radical, primary root, and shoot length, compared to WT seedlings. We further found that OsCUL1 proteins, particularly OsCUL1-1 and OsCUL1-3, interact with OsMBTB32 and may suppress the function of OsMBTB32 during salt stress. Moreover, OsWRKY42, a homolog of ZmWRKY114 which negatively regulates salt stress in rice, directly binds to the W-box of OsCUL1-1 and OsCUL1-3 promoters to promote the interaction of OsCUL1-1 and OsCUL1-3 with OsMBTB32 protein in rice. The overexpression of OsMBTB32 and OsCUL1-3 further confirmed the function of OsMBTB32 and OsCUL1s in salt tolerance in Arabidopsis. Overall, the findings of the present study provide promising knowledge regarding the MATH-BTB domain-containing proteins and their role in enhancing the growth and development of rice under salt stress.MATH-BTB proteins are involved in a variety of cellular processes that regulate cell homeostasis and developmental processes. Previous studies reported the involvement of BTB proteins in the development of various organs in plants; however, the function of BTB proteins in salt stress is less studied. Here, we found a novel MATH-BTB domain-containing OsMBTB32 protein that was highly expressed in leaf, root, and shoot. The up-regulation of the OsMBTB32 transcript in 2-week-old seedlings under salt stress suggests the significant role of the OsMBTB32 gene in salinity. The OsMBTB32 transgenic seedlings (OE and RNAi) exhibited significant differences in various phenotypes, including plumule, radical, primary root, and shoot length, compared to WT seedlings. We further found that OsCUL1 proteins, particularly OsCUL1-1 and OsCUL1-3, interact with OsMBTB32 and may suppress the function of OsMBTB32 during salt stress. Moreover, OsWRKY42, a homolog of ZmWRKY114 which negatively regulates salt stress in rice, directly binds to the W-box of OsCUL1-1 and OsCUL1-3 promoters to promote the interaction of OsCUL1-1 and OsCUL1-3 with OsMBTB32 protein in rice. The overexpression of OsMBTB32 and OsCUL1-3 further confirmed the function of OsMBTB32 and OsCUL1s in salt tolerance in Arabidopsis. Overall, the findings of the present study provide promising knowledge regarding the MATH-BTB domain-containing proteins and their role in enhancing the growth and development of rice under salt stress.

Metabolic resistance to acetolactate synthase inhibitors in Beckmannia syzigachne: identification of CYP81Q32 and its transcription regulation.

Frequent herbicide use selects for herbicide resistance in weeds. Cytochrome P450s are important detoxification enzymes responsible for herbicide resistance in plants. We identified and characterized a candidate P450 gene (BsCYP81Q32) from the problematic weed Beckmannia syzigachne to test whether it conferred metabolic resistance to the acetolactate synthase-inhibiting herbicides mesosulfuron-methyl, bispyribac-sodium, and pyriminobac-methyl. Transgenic rice overexpressing BsCYP81Q32 was resistant to the three herbicides. Equally, rice overexpressing the rice ortholog gene OsCYP81Q32 was more resistant to mesosulfuron-methyl. Conversely, an OsCYP81Q32 gene knockout generated using CRISPR/Cas9 enhanced mesosulfuron-methyl sensitivity in rice. Overexpression of the BsCYP81Q32 gene resulted in enhanced mesosulfuron-methyl metabolism in transgenic rice seedlings via O-demethylation. The major metabolite, demethylated mesosulfuron-methyl, was chemically synthesized and displayed reduced herbicidal effect in plants. Moreover, a transcription factor (BsTGAL6) was identified and shown to bind a key region in the BsCYP81Q32 promoter for gene activation. Inhibition of BsTGAL6 expression by salicylic acid treatment in B. syzigachne plants reduced BsCYP81Q32 expression and consequently changed the whole plant response to mesosulfuron-methyl. Sequence polymorphisms in an important region of the BsTGAL6 promoter may explain the higher expression of BsTGAL6 in resistant versus susceptible B. syzigachne plants. Collectively, the present study reveals the evolution of an herbicide-metabolizing and resistance-endowing P450 and its transcription regulation in an economically important weedy plant species.

Strigolactones in Sugarcane Growth and Development

Sugarcane is a complex polyploid aneuploid cash crop, and transgenic varieties are important for molecular genetic and traditional breeding approaches. Herein, the sugarcane variety ROC22 served as the receptor, the Bar gene served as a screening marker, and positive and negative fragments of the ScD27.2 gene, upstream of strigolactones (SLs) biosynthesis genes driven by the 35S promoter, were introduced by Agrobacterium tumefaciens-mediated transformation. Regenerated plants were obtained by co-culture, screening culture, and differentiation induction, and 27 sense and antisense ScD27.2 transgenic seedlings were obtained by herbicide screening. PCR detection and 1% Basta (Glufosinate) application on leaves revealed Bar in all lines, with all testing positive for herbicide application and 23 containing the target gene (positive resistance screening rate = 87.5%). q-PCR and phenotypic analyses showed that ScD27.2 expression, plant height, tiller number, root length, stem diameter, and fresh weight were decreased in transgenic (ScD27.2R-9) compared with non-transgenic (NT and ScD27.2F-2) lines. ScD27.2 expression was downregulated, and growth potential was inhibited. Under 20% PEG treatment, malondialdehyde (MDA) content in ScD27.2R-9 was higher than in NT, while proline content was lower. Under drought stress, ScD27.2 expression, MDA levels, and proline content in ScD27.2F-2 and NT were higher than in non-treated controls, ScD27.2 expression increased with time, and MDA and proline levels also increased. ScD27.2 expression in ScD27.2R-9 decreased under 20% PEG treatment, MDA and proline increased (but not to NT levels), and growth was lower than NT. The 20% PEG treatment also increased the levels of (±)-2′-epi-5-deoxystrigol and (+)-abscisic acid in the rooting culture media of ScD27.2F-2, ScD27.2R-9, and NT lines, but the levels of (+)-abscisic acid content in ScD27.2R-9 was lower than in NT. Thus, interfering with ScD27.2 expression decreased resistance to 20% PEG treatment. ScD27.2 encodes a β-carotene isomerase involved in SLs biosynthesis that might function in sugarcane resistance to drought stress. It explains the role of SLs in sugarcane growth and development and responses to drought stress.

The cotton protein GhIQD21 interacts with GhCaM7 and modulates organ morphogenesis in Arabidopsis by influencing microtubule stability.

GhIQD21, a cotton IQ67-domain protein, interacts with GhCaM7 and alters organ shape in Arabidopsis by modulating microtubule stability. Calcium ion (Ca2+) and the calcium sensor calmodulin play crucial roles in the growth and development of plants. GhCaM7, a calmodulin in upland cotton (Gossypium hirsutum L.), is highly expressed in cotton fiber cells during the rapid elongation period and plays an important role in fiber cell development. In this study, we screened for GhCaM7-interacting proteins and identified GhIQD21, which contains a typical IQ67-domain. GhIQD21 was preferentially expressed at the fiber rapid elongation stage, and the protein localized to microtubules (MTs). Ectopic expression of GhIQD21 in Arabidopsis resulted in shorter leaves, petals, siliques, and plant height, thicker inflorescences, and more trichomes when compared with wild type (WT). Further investigation indicated that the morphogenesis of leaf epidermal cells and silique cells was altered. There was less consistency in the orientation of cortical microtubules in cotyledon and hypocotyl epidermal cells. Furthermore, compared with WT, transgenic seedling hypocotyls were more sensitive to oryzalin, a MT depolymerization drug. These results indicated that GhIQD21 is a GhCaM7-interacting protein located in MTs and that it plays a role in plant growth and potentially cotton fiber development. This study provides a foundation for further studies of the function and regulatory mechanism of GhIQD21 in fiber cell development.

Overexpression of GmNF-YA14 produced multiple phenotypes in soybean

Soybean is an important oil crop, and more and more soybean gene functions have been reported; however, one soybean gene with multiple functions is rarely seen. The NF-YA subfamily plays a broad regulatory role in plant growth and development, and GmNF-YA14 (Glyma.14G010000) is one of them. We found that the expression level of GmNF-YA14 in the root of soybean seedlings was significantly increased after a one-hour treatment with 200 mM salt. The transgenic soybean was obtained by Agrobacterium-mediated method, and all experiments were carried out using transgenic soybean homozygous lines. Under a 200 mM salt treatment, the leaf chlorophyll content of overexpressed GmNF-YA14 transgenic soybean seedlings was significantly decreased compared to the control. Moreover, the first flowering time of overexpressed GmNF-YA14 transgenic soybean was shortened by more than six days compared to the control, and the growth period structure changed significantly. Overexpressed GmNF-YA14 transgenic soybean plants had two or three branches, whereas control soybean plants had eight or nine branches. Compared to the control, the number of main stem nodes and plant height of overexpressed GmNF-YA14 transgenic soybean plants were significantly reduced. The 100-seed weight of GmNF-YA14 transgenic soybean line OE-8 was significantly higher than that of the control. Transcriptome analysis indicated that the multiple phenotypes of the overexpressed GmNF-YA14 transgenic soybean may be related to the mitogen-activated protein kinase (MAPK) signaling pathway. These results indicated that compared to the control, overexpressed GmNF-YA14 transgenic soybeans had changes in salt stress tolerance, growth period, plant type, and 100-seed weight, and these changes may be related to the MAPK signaling pathway.

Dehydrin AmDH1 improves drought tolerance in transgenic maize plants

Dehydrin is a large protein group to have protective functions to plant cells under osmotic stress. Overexpression of dehydrin encoded genes isolated from mangrove plants had improved drought tolerance of transgenic plants. Therefore, in this research, a coding sequence with the length of 573bp that encodes Dehydrin1 (DH1) protein was isolated from a mangrove plant namely Mam bien (Avicennia marina) which naturally grows in Xuan Thuy national park, Vietnam. The coding sequence was denoted as AmDH1 and cloned into the plant transformation vector pCAM 35S-AmDH1. The transgenic vector was transformed into A. tumefiaciens cells and the vector was transferred into the K7 maize line to generate transgenic maize seedlings. The AmDH1 transgenic maize T2 seedlings have shown an improved drought tolerance than the original K7 maize line.

Overexpression of OsERF106MZ promotes parental root growth in rice seedlings by relieving the ABA-mediated inhibition of root growth under salinity stress conditions

BackgroundRoots are essential for plant growth and have a variety of functions, such as anchoring the plant to the ground, absorbing water and nutrients from the soil, and sensing abiotic stresses, among others. OsERF106MZ is a salinity-induced gene that is expressed in germinating seeds and rice seedling roots. However, the roles of OsERF106MZ in root growth remain poorly understood.ResultsHistochemical staining to examine β-glucuronidase (GUS) activity in transgenic rice seedlings harboring OsERF106MZp::GUS indicated that OsERF106MZ is mainly expressed in the root exodermis, sclerenchyma layer, and vascular system. OsERF106MZ overexpression in rice seedlings leads to an increase in primary root (PR) length. The phytohormone abscisic acid (ABA) is thought to act as a hidden architect of root system structure. The expression of the ABA biosynthetic gene OsAO3 is downregulated in OsERF106MZ-overexpressing roots under normal conditions, while the expression of OsNPC3, an AtNPC4 homolog involved in ABA sensitivity, is reduced in OsERF106MZ-overexpressing roots under both normal and NaCl-treated conditions. Under normal conditions, OsERF106MZ-overexpressing roots show a significantly reduced ABA level; moreover, exogenous application of 1.0 µM ABA can suppress OsERF106MZ-mediated root growth promotion. Additionally, OsERF106MZ-overexpressing roots display less sensitivity to ABA-mediated root growth inhibition when treated with 5.0 µM ABA under normal conditions or exposed to NaCl-treated conditions. Furthermore, chromatin immunoprecipitation (ChIP)-qPCR and luciferase (LUC) reporter assays showed that OsERF106MZ can bind directly to the sequence containing the GCC box in the promoter region of the OsAO3 gene and repress the expression of OsAO3.ConclusionsOsERF106MZ may play a role in maintaining root growth for resource uptake when rice seeds germinate under salinity stress by alleviating ABA-mediated root growth inhibition.

Expression of the Arabidopsis Mg-chelatase H subunit alleviates iron deficiency-induced stress in transgenic rice.

The most common symptom of iron (Fe) deficiency in plants is leaf chlorosis caused by impairment of chlorophyll biosynthesis. Magnesium (Mg)-chelatase H subunit (CHLH) is a key component in both chlorophyll biosynthesis and plastid signaling, but its role in Fe deficiency is poorly understood. Heterologous expression of the Arabidopsis thaliana Mg-chelatase H subunit gene (AtCHLH) increased Mg-chelatase activity by up to 6-fold and abundance of its product, Mg-protoporphyrin IX (Mg-Proto IX), by 60-75% in transgenic rice (Oryza sativa) seedlings compared to wild-type (WT) controls. Noticeably, the transgenic seedlings showed alleviation of Fe deficiency symptoms, as evidenced by their less pronounced leaf chlorosis and lower declines in shoot growth, chlorophyll contents, and photosynthetic efficiency, as indicated by F v/F m and electron transport rate, compared to those in WT seedlings under Fe deficiency. Porphyrin metabolism was differentially regulated by Fe deficiency between WT and transgenic seedlings, particularly with a higher level of Mg-Proto IX in transgenic lines, showing that overexpression of AtCHLH reprograms porphyrin metabolism in transgenic rice. Leaves of Fe-deficient transgenic seedlings exhibited greater upregulation of deoxymugineic acid biosynthesis-related genes (i.e., NAS, NAS2, and NAAT1), YSL2 transporter gene, and Fe-related transcription factor genes IRO2 and IDEF2 than those of WT, which may also partly contribute to alleviating Fe deficiency. Although AtCHLH was postulated to act as a receptor for abscisic acid (ABA), exogenous ABA did not alter the phenotypes of Fe-deficient WT or transgenic seedlings. Our study demonstrates that modulation of porphyrin biosynthesis through expression of AtCHLH in transgenic rice alleviates Fe deficiency-induced stress, suggesting a possible role for CHLH in Fe deficiency responses.

Sucrose non-fermenting1-related protein kinase VcSnRK2.3 promotes anthocyanin biosynthesis in association with VcMYB1 in blueberry.

Sucrose non-fermenting1-related protein kinase-2 (SnRK2) is a plant-specific protein kinase family and an important component of the abscisic acid (ABA) signaling pathway. However, there is a lack of relevant studies in blueberry (Vaccinium corymbosum). In this study, we identified six SnRK2 family members (from VcSnRK2.1 to VcSnRK2.6) in blueberries for the first time. In addition, we found that VcSnRK2.3 expression was not only positively correlated with fruit ripening but was also induced by ABA signaling. Transient expression in blueberry fruits also proved that VcSnRK2.3 promoted anthocyanin accumulation and the expression of anthocyanin synthesis-related genes such as VcF3H, VcDFR, VcANS, and VcUFGT. Transgenic Arabidopsis thaliana seeds and seedlings overexpressing VcSnRK2.3 showed anthocyanin pigmentation. Yeast two-hybrid assays (Y2H) and Bimolecular fluorescence complementation assays (BiFC) demonstrated that VcSnRK2.3 could interact with the anthocyanin positive regulator VcMYB1. Finally, VcSnRK2.3 was able to enhance the binding of VcMYB1 to the VcDFR promoter. Via regulation transcription of anthocyanin biosynthesis genes, VcSnRK2.3 promoted anthocyanin accumulation in blueberry. The above results suggest that VcSnRK2.3 plays an important role in blueberry anthocyanin synthesis, is induced by ABA, and can interact with VcMYB1 to promote anthocyanin biosynthesis in blueberry.

Overexpression of ZmDUF1644 from Zoysia matrella enhances salt tolerance in Arabidopsis thaliana

The DUF1644 family is a highly conserved and functionally unknown protein family and it has been demonstrated that it significantly improved the salt tolerance of rice (non-halophytes). However, little is understood about the function of the DUF1644 gene in halophytes. This study aimed to investigate whether the DUF1644 gene has a similar function in non-halophytes as in halophytic species. The functions of ZmDUF1644 transferred from the halophytic Zoysia matrella into non-halophytic Arabidopsis thaliana were investigated under salt stress (150 mM NaCl). Phylogenetic analysis clustered ZmDUF1644 and OsSDIP361 in the same group, which function as salt-tolerant genes in rice. The obtained results exhibited that the ZmDUF1644 gene significantly alleviated the adverse effects of salt stress on transgenic Arabidopsis thaliana seedlings. The maximum root length, fresh weight, and photosynthetic pigment content of the transgenic seedlings were obviously better than that of the wild type. The relative electrolyte leakage rate, Na+ content, and Na+/K+ ratios were relatively lower in the transgenic line than in the wild type under salt stress. Moreover, the expression of AtSOS2 gene was also significantly higher than that of wild-type under salt stress. The study concluded that the ZmDUF1644 gene was significantly overexpressed in salt-stressed Arabidopsis thaliana, enhancing its salt tolerance. It is the first report on the functions of ZmDUF1644 from halophytic Zoysia matrella which has a similar function in glycophytic species or non-halophytic species.

Genome-wide identification of DnaJ gene family in Catalpa bungei and functional analysis of CbuDnaJ49 in leaf color formation.

DnaJs are the common molecular chaperone proteins with strong structural and functional diversity. In recent years, only several DnaJ family members have been found to be able to regulate leaf color, and it remains to be explored whether there are other potential members that also regulate this character. Here, we identified 88 putative DnaJ proteins from Catalpa bungei, and classified them into four types according to their domain. Gene-structure analysis revealed that each member of CbuDnaJ family had same or similar exon-intron structure. Chromosome mapping and collinearity analysis showed that tandem and fragment duplication occurred in the process of evolution. Promoter analyses suggested that CbuDnaJs might be involved in a variety of biological processes. The expression levels of DnaJ family members in different color leaves of Maiyuanjinqiu were respectively extracted from the differential transcriptome. Among these, CbuDnaJ49 was the largest differentially expressed gene between the green and yellow sectors. Ectopic overexpression of CbuDnaJ49 in tobacco showed that the positive transgenic seedlings exhibited albino leaves, and the contents of chlorophyll and carotenoid were significantly reduced compared with those of wild type. The results suggested that CbuDnaJ49 played an important role in regulating leaf color. This study not only identified a novel gene of DnaJ family members regulating leaf color, but also provided new germplasm for landscaping.

Genome-wide characterization of the U-box gene in Camellia sinensis and functional analysis in transgenic tobacco under abiotic stresses

Plants U-box genes are crucial for plant survival, and they extensively regulate plant growth, reproduction and development as well as coping with stress and other processes. In this study, we identified 92 CsU-box genes through genome-wide analysis in the tea plant (Camellia sinensis), all of them contained the conserved U-box domain and were divided into 5 groups, which supported by the further genes structure analysis. The expression profiles in eight tea plant tissues and under abiotic and hormone stresses were analyzed using the TPIA database. 7 CsU-box genes (CsU-box27/28/39/46/63/70/91) were selected to verify and analyze expression patterns under PEG-induced drought and heat stress in tea plant respectively, the qRT-PCR results showed consistent with transcriptome datasets; and the CsU-box39 were further heterologous expressed in tobacco to perform gene function analysis. Phenotypic analyses of overexpression transgenic tobacco seedlings and physiological experiments revealed that CsU-box39 positively regulated the plant response to drought stress. These results lay a solid foundation for studying the biological function of CsU-box, and will provide breeding strategy basis for tea plant breeders.

ASR1 and ASR2, Two Closely Related ABA-Induced Serine-Rich Transcription Repressors, Function Redundantly to Regulate ABA Responses in Arabidopsis.

The plant hormone abscisic acid (ABA) is able to regulate the expression of ABA-responsive genes via signaling transduction, and thus plays an important role in regulating plant responses to abiotic stresses. Hence, characterization of unknown ABA response genes may enable us to identify novel regulators of ABA and abiotic stress responses. By using RT-PCR analysis, we found that the expression levels of ABA-induced Serine-rich Repressor 1 (ASR1)and ASR2, two closely related unknown function genes, were increased in response to ABA treatment. Amino acid sequence analyses show that ASR1 contains an L×L×L motif and both ASR1 and ASR2 are enriched in serine. Transfection assays in Arabidopsis leaf protoplasts show that ASR1 and ASR2 were predominantly localized in the nucleus and were able to repress the expression of the reporter gene. The roles of ASRs in regulating ABA responses were examined by generating transgenic Arabidopsis plants over-expressing ASR1 and ASR2, respectively, and CRISPR/Cas9 gene-edited single and double mutants for ASR1 and ASR2. In both the seed germination and cotyledon greening assays, ABA sensitivity remained largely unchanged in the over-expression transgenic plants and the single mutants of ASR1 and ASR2, but greatly increased ABA sensitivity was observed in the asr1 asr2 double mutants. In root elongation assays, however, decreased ABA sensitivity was observed in the 35S:ASR1 and 35S:ASR2 transgenic plants, whereas increased ABA sensitivity was observed in the asr1 and asr2 single mutants, and ABA sensitivity was further increased in the asr1 asr2 double mutants. Transcriptome analysis show that the differentially expressed genes (DEGs) down-regulated in the 35S:ASR1 transgenic plant seedlings, but up-regulated in the asr1 asr2 double mutant seedlings were highly enriched in processes including responses to plant hormones and stress stimuli. Taken together, our results show that ASR1 and ASR2 are closely related ABA response genes, ASR1 and ASR2 are serine-rich novel transcription repressors, and they negatively regulate ABA responses in Arabidopsis in a redundant manner.

Wheat Selenium-binding protein TaSBP-A enhances cadmium tolerance by decreasing free Cd2+ and alleviating the oxidative damage and photosynthesis impairment.

Cadmium, one of the toxic heavy metals, robustly impact crop growth and development and food safety. In this study, the mechanisms of wheat (Triticum aestivum L.) selenium-binding protein-A (TaSBP-A) involved in response to Cd stress was fully investigated by overexpression in Arabidopsis and wheat. As a cytoplasm protein, TaSBP-A showed a high expression in plant roots and its expression levels were highly induced by Cd treatment. The overexpression of TaSBP-A enhanced Cd-toleration in yeast, Arabidopsis and wheat. Meanwhile, transgenic Arabidopsis under Cd stress showed a lower H2O2 and malondialdehyde content and a higher photochemical efficiency in the leaf and a reduction of free Cd2+ in the root. Transgenic wheat seedlings of TaSBP exhibited an increment of Cd content in the root, and a reduction Cd content in the leaf under Cd2+ stress. Cd2+ binding assay combined with a thermodynamics survey and secondary structure analysis indicated that the unique CXXC motif in TaSBP was a major Cd-binding site participating in the Cd detoxification. These results suggested that TaSBP-A can enhance the sequestration of free Cd2+ in root and inhibit the Cd transfer from root to leaf, ultimately conferring plant Cd-tolerance via alleviating the oxidative stress and photosynthesis impairment triggered by Cd stress.

Comprehensive analysis of cucumber RAV family genes and functional characterization of CsRAV1 in salt and ABA tolerance in cucumber.

The RAV (related to ABI3 and VP1) transcription factors are specific and exist in plants, which contain a B3 DNA binding domain and/or an APETALA2 (AP2) DNA binding domain. RAVs have been extensively studied in plants, and more and more evidences show that RAVs are involved in various aspects of plant growth and development, stress resistance and hormone signal transduction. However, the systematic analysis of RAV family in cucumber is rarely reported. In this study, eight CsRAV genes were identified in cucumber genome and we further comprehensively analyzed their protein physicochemical properties, conserved domains, gene structure and phylogenetic relationships. The synteny analysis and gene duplications of CsRAV genes were also analysed. Cis-element analysis revealed that the CsRAVs promoter contained several elements related to plant hormones and abiotic stress. Expression analysis showed that NaCl and ABA could significantly induce CsRAV genes expression. Subcellular localization revealed that all CsRAVs were localized in the nucleus. In addition, 35S:CsRAV1 transgenic Arabidopsis and cucumber seedlings enhanced NaCl and ABA tolerance, revealing CsRAV1 may be an important regulator of abiotic stress response. In conclusion, comprehensive analysis of CsRAVs would provide certain reference for understanding the evolution and function of the CsRAV genes.

Identifying a Detoxifying Uridine Diphosphate Glucosyltransferase (UGT), MdUGT83K2, Which Can Glycosylate the Aryloxyphenoxypropionate Herbicide

Glycosylation is a common modification reaction in plants. The products obtained upon glycosylation have different biological functions, making glycosylation an important mechanism affecting and regulating the balance of plant growth and metabolism. In this study, we first speculated that Group I in the apple glycosyltransferase family may have a predicted function like UGT83A1, according to gene chip data published online. Subsequently, by real-time PCR (polymerase chain reaction), we analyzed whether the expression of nine glycosyltransferase genes in Group I was induced by our previously reported ACCase (Acetyl-CoA carboxylase) inhibition-based herbicide QPP ((R)-ethyl·2-(4-((6-fluoro-3-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)oxy) phenoxy) propanoate). It was found that expression of the MdUGT83K2 gene in Group I was significantly increased by QPP. In order to determine whether MdUGT83K2 can glycosylate QPP, we confirmed the enzymatic reaction of MdUGT83K2 in vitro and the presence of QPP glycosides in MdUGT83K2 transgenic apple seedlings by HPLC (High Performance Liquid Chromatography), and found that MdUGT83K2 can transfer glucose to QPP in vivo, which is glycosylated. In this work, we identified a novel apple glycosyltransferase, MdUGT83K2, which functions to glycosylate the ACCase-inhibiting herbicide QPP and may be involved in plant detoxification. Key Contribution: A novel apple glycosyltransferase, MdUGT83K2, was identified, which may be involved in plant detoxification by glycosylation modification of the ACCase-inhibiting herbicide.

BcAMT1;5 Mediates Nitrogen Uptake and Assimilation in Flowering Chinese Cabbage and Improves Plant Growth When Overexpressed in Arabidopsis

Nitrogen (N) is a major limiting factor for plant growth and vegetable production. Understanding the regulatory mechanisms of N uptake, transport, and assimilation is key to improving N use efficiency in plants. Ammonium transporters (AMTs) play an important role in plant N metabolism. In this study, we isolated an important AMT1 subfamily member (BcAMT1;5) with a highly conserved signatural AMT1 subfamily motif from flowering Chinese cabbage. Based on functional complementation in yeast mutant 31019b and overexpression of BcAMT1;5 in Arabidopsis, BcAMT1;5 is a functional AMT. Tissue expression analysis showed that BcAMT1;5 was mainly expressed in roots and showed multiple N regime transcript patterns to respond to varying nutritional conditions. This was up-regulated by N-deficiency and down-regulated by supplying NH4+. The glucuronidase (GUS) activities of BcAMT1;5pro::GUS showed a similar change in response to different N conditions. Overexpression of BcAMT1;5 accelerated the growth of transgenic seedlings, increased NH4+ net influxes, and enhanced the content and accumulation of NH4+ and NO3− at low N concentrations. Additionally, it increased the transcript levels of N assimilation-related genes in shoots. These results indicate that BcAMT1;5 may participate in N uptake and assimilation under various N conditions in flowering Chinese cabbage, but it was differed obviously from other AMT1s.