Multigene manipulation of photosynthetic carbon metabolism enhances the photosynthetic capacity and biomass yield of cucumber under low-CO2 environment.

Solar greenhouses are important in the vegetable production and widely used for the counter-season production in the world. However, the CO2 consumed by crops for photosynthesis after sunrise is not supplemented and becomes chronically deficient due to the airtight structure of solar greenhouses. Vegetable crops cannot effectively utilize light resources under low-CO2 environment, and this incapability results in reduced photosynthetic efficiency and crop yield. We used cucumber as a model plant and generated several sets of transgenic cucumber plants overexpressing individual genes, including ß-carbonic anhydrase 1 (CsßCA1), ß-carbonic anhydrase 4 (CsßCA4), and sedoheptulose-1,7-bisphosphatase (CsSBP); fructose-1,6-bisphosphate aldolase (CsFBA), and CsßCA1 co-expressing plants; CsßCA4, CsSBP, and CsFBA co-expressing plants (14SF). The results showed that the overexpression of CsßCA1, CsßCA4, and 14SF exhibited higher photosynthetic and biomass yield in transgenic cucumber plants under low-CO2 environment. Further enhancements in photosynthesis and biomass yield were observed in 14SF transgenic plants under low-CO2 environment. The net photosynthesis biomass yield and photosynthetic rate increased by 49% and 79% compared with those of the WT. However, the transgenic cucumbers of overexpressing CsFBA and CsSBP showed insignificant differences in photosynthesis and biomass yield compared with the WT under low-CO2.environment. Photosynthesis, fluorescence parameters, and enzymatic measurements indicated that CsßCA1, CsßCA4, CsSBP, and CsFBA had cumulative effects in photosynthetic carbon assimilation under low-CO2 environment. Co-expression of this four genes (CsßCA1, CsßCA4, CsSBP, and CsFBA) can increase the carboxylation activity of RuBisCO and promote the regeneration of RuBP. As a result, the 14SF transgenic plants showed a higher net photosynthetic rate and biomass yield even under low-CO2environment.These findings demonstrate the possibility of cultivating crops with high photosynthetic efficiency by manipulating genes involved in the photosynthetic carbon assimilation metabolic pathway.

The Accumulation of Lutein and ß-Carotene and Transcript Profiling of Genes Related to Carotenoids Biosynthesis in Yellow Celery.

Carotenoids are the general term of natural pigments. The formation of plant color is probably related to the components of carotenoids. As the yellow variety of celery, it is rich in the composition and content of carotenoids. However, the transcript profiling and roles of the genes related to carotenoids biosynthesis in yellow celery remain unclear. In this study, three yellow celery cultivars at different growth stages were used to analyze the content and composition of carotenoids and transcriptional changes of carotenoid biosynthesis-related genes. The lutein and ß-carotene were detected in yellow celery cultivar, while α-carotene and lycopene were not detected. The contents of lutein and ß-carotene were higher in leaf blades than in petioles. During the growth and development, the contents of lutein and ß-carotene gradually decreased in celery. Compared with the other two cultivars, the contents of lutein and ß-carotene were the highest in 'Huangtaiji' of 65 days after sowing (DAS) and 85 DAS and 'Liuhehuangxinqin' of 105 DAS, respectively. The expression levels of AgLCYB and AgPSY2 genes were significantly correlated with lutein and ß-carotene contents. This work provided a reference for the further study on carotenoid metabolisms in yellow celery and also made sense on the way of cultivating yellow celery with high carotenoids content.

The gene encoding lycopene epsilon cyclase of celery enhanced lutein and ß-carotene contents and confers increased salt tolerance in Arabidopsis.

Celery (Apium graveolens L.) is a leafy vegetable of Apiaceae, which is greatly popular because of its rich nutrients. Lutein and ß-carotene are two important carotenoids. Lycopene epsilon cyclase (LCY-ε) is a key branch point enzyme in the carotenoid biosynthetic pathway. In this study, we cloned the AgLCY-ε gene from celery and overexpressed it in Arabidopsis. The results showed that both lutein and ß-carotene accumulation increased significantly in transgenic Arabidopsis hosting AgLCY-ε gene, compared with wild type (WT) plants. The transcription levels of AtPSY and AtCRTISO genes involved in carotenoids biosynthesis also increased in transgenic lines. One-month-old transgenic Arabidopsis seedlings were treated with 200 mM NaCl. The malondialdehyde (MDA) content in transgenic Arabidopsis plants after salt treatment was significantly lower, and the activities of the two antioxidant enzymes, superoxide dismutase (SOD) and peroxidase (POD), were significantly increased than that of WT plants. Overexpression of AgLCY-ε gene showed increased lutein and ß-carotene accumulations, and enhanced salt tolerance in transgenic plants.

A novel plant protein-disulfide isomerase participates in resistance response against the TYLCV in tomato.

MAIN CONCLUSION: Overexpression or silencing of the SlPDI could increase plants resistance or sensitivity to TYLCV through enhancing or reducing the plant's antioxidant capacity. Tomato yellow leaf curl virus (TYLCV), a plant virus that could infect a variety of crops, is particularly destructive to tomato growth. Protein disulfide isomerase (PDI) is a member of the thioredoxin (Trx) superfamily, is capable of catalyzing the formation and heterogeneity of protein disulfide bonds and inhibiting the aggregation of misfolded proteins. Studies have shown that PDI plays important roles in plant response to abiotic stress, there is no research report on the function of PDI in response to biotic stress, especially TYLCV infection. Here, we identified a tomato PDI gene, SlPDI, was involved in regulating tomato plants resistance to TYLCV. Subcellular localization results showed that SlPDI was located at the endoplasmic reticulum (ER), and its location remained unchanged after infection with TYLCV virus. Overexpression or silencing of SlPDI could increase plants resistance or sensitivity to TYLCV. Transgenic plants that overexpressing SlPDI exhibit enhanced antioxidant activity evidenced by lower hydrogen peroxide (H2O2) level and higher activity of superoxide dismutase (SOD) and peroxidase (POD) in comparison with WT plants, after infected by TYLCV. Moreover, the SlPDI-silencing plants showed opposite results. The promoter analyzes result showed that SlPDI was involved in response to salicylic acid (SA), and our experimental results also showed that the expression level of SlPDI was induced by SA. Taken together, our results indicated that SlPDI could regulate plant resistance to TYLCV through enhancing the protein folding function of ER and promoting the synthesis and conformation of antioxidant-related proteins.

Advances in AP2/ERF super-family transcription factors in plant.

In the whole life process, many factors including external and internal factors affect plant growth and development. The morphogenesis, growth, and development of plants are controlled by genetic elements and are influenced by environmental stress. Transcription factors contain one or more specific DNA-binding domains, which are essential in the whole life cycle of higher plants. The AP2/ERF (APETALA2/ethylene-responsive element binding factors) transcription factors are a large group of factors that are mainly found in plants. The transcription factors of this family serve as important regulators in many biological and physiological processes, such as plant morphogenesis, responsive mechanisms to various stresses, hormone signal transduction, and metabolite regulation. In this review, we summarized the advances in identification, classification, function, regulatory mechanisms, and the evolution of AP2/ERF transcription factors in plants. AP2/ERF family factors are mainly classified into four major subfamilies: DREB (Dehydration Responsive Element-Binding), ERF (Ethylene-Responsive-Element-Binding protein), AP2 (APETALA2) and RAV (Related to ABI3/VP), and Soloists (few unclassified factors). The review summarized the reports about multiple regulatory functions of AP2/ERF transcription factors in plants. In addition to growth regulation and stress responses, the regulatory functions of AP2/ERF in plant metabolite biosynthesis have been described. We also discussed the roles of AP2/ERF transcription factors in different phytohormone-mediated signaling pathways in plants. Genomic-wide analysis indicated that AP2/ERF transcription factors were highly conserved during plant evolution. Some public databases containing the information of AP2/ERF have been introduced. The studies of AP2/ERF factors will provide important bases for plant regulatory mechanisms and molecular breeding.

Selection of appropriate reference genes for RT-qPCR analysis under abiotic stress and hormone treatment in celery.

Celery is one of the most important vegetable crop and its yield and quality is influenced by many environmental factors. Researches on gene expression not only help to unravel the molecular regulatory mechanism but also identify the key genes in the biological response. RT-qPCR is a commonly used technology to quantify the gene expression. Selecting an appropriate reference gene is an effective approach to improve the accuracy of RT-qPCR assay. To our knowledge, the evaluation of reference genes under different treatments in celery has not been reported yet. In this study, the expression stabilities of eight candidate reference genes (ACTIN, eIF-4α , GAPDH, TBP, TUB-A, UBC, TUB-B, and EF-1α ) under abiotic stresses (heat, cold, drought, and salt) and hormone treatments (SA, MeJA, GA, and ABA) were detected. The expression stabilities of candidate genes were compared and ranked by geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder programs. The results calculated by different programs were not completely consistent. Considering the comprehensive analysis results, ACTIN was the most stable reference gene and TUB-B showed the worst expression stabilities under the selected abiotic stress and hormone treatments in celery. The reliability of reference genes was further confirmed by the normalization of CAT1 gene under drought stress. This study presented evidences and basis to select the appropriate reference genes under different treatments in celery.

Introduction of Exogenous Glycolate Catabolic Pathway Can Strongly Enhances Photosynthesis and Biomass Yield of Cucumber Grown in a Low-CO2 Environment.

Carbon dioxide (CO2) is very important for photosynthesis of green plants. CO2 concentration in the atmosphere is relatively stable, but it drops sharply after sunrise due to the tightness of the greenhouse and the absorption of CO2 by vegetable crops. Vegetables in greenhouses are chronically CO2 starved. To investigate the feasibility of using genetic engineering to improve the photosynthesis and yield of greenhouse cucumber in a low CO2 environment, five genes encoding glyoxylate carboligase (GCL), tartronic semialdehyde reductase (TSR), and glycolate dehydrogenase (GlcDH) in the glycolate catabolic pathway of Escherichia coli were partially or completely introduced into cucumber chloroplast. Both partial pathway by introducing GlcDH and full pathway expressing lines exhibited higher photosynthetic efficiency and biomass yield than wild-type (WT) controls in low CO2 environments. Expression of partial pathway by introducing GlcDH increased net photosynthesis by 14.9% and biomass yield by 44.9%, whereas the expression of the full pathway increased seed yield by 33.4% and biomass yield by 59.0%. Photosynthesis, fluorescence parameters, and enzymatic measurements confirmed that the introduction of glycolate catabolic pathway increased the activity of photosynthetic carbon assimilation-related enzymes and reduced the activity of photorespiration-related enzymes in cucumber, thereby promoting the operation of Calvin cycle and resulting in higher net photosynthetic rate even in low CO2 environments. This increase shows an improvement in the efficiency of the operation of the photosynthetic loop. However, the utilization of cucumber of low concentration CO2 was not alleviated. This study demonstrated the feasibility of introducing the pathway of exogenous glycolate catabolic pathway to improve the photosynthetic and bio-yield of cucumber in a low CO2 environment. These findings are of great significance for high photosynthetic efficiency breeding of greenhouse cucumber.

DcC4H and DcPER Are Important in Dynamic Changes of Lignin Content in Carrot Roots under Elevated Carbon Dioxide Stress.

In our study, isobaric tags for relative and absolute quantification (iTRAQ) was conducted to determine the significantly changed proteins in the fleshy roots of carrots under different carbon dioxide (CO2) treatments. A total of 1523 proteins were identified, of which 257 were differentially expressed proteins (DEPs). On the basis of annotation analysis, the DEPs were identified to be involved in energy metabolism, carbohydrate metabolism, and some other metabolic processes. DcC4H and DcPER, two lignin-related proteins, were identified from the DEPs. Under elevated CO2 stress, both carrot lignin content and the expression profiles of lignin biosynthesis genes changed significantly. The protein-protein interactions among lignin-related enzymes proved the importance of DcC4H and DcPER. The results of our study provided potential new insights into the molecular mechanism of lignin content changes in carrot roots under elevated CO2 stress.

AP2/ERF Transcription Factors Involved in Response to Tomato Yellow Leaf Curly Virus in Tomato.

Tomato yellow leaf curly virus (TYLCV), transmitted by the whitefly (), causes leaf curling and yellowing, plant dwarfism, and growth inhibition in tomato ( L.). The APETALA2 (AP2) and ethylene response factor (ERF) transcription factor (TF) family, the largest plant-specific TF family, was identified to function in plant development and pathogen defense. Our study aimed to analyze the mechanism underlying the function of ERF (SlERF) TFs in response to TYLCV infection and improve useful information to increase the resistance to TYLCV in tomato. A total of 22 tomato AP2/ERF TFs in response to TYLCV were identified according to transcriptome database. Five ERF-B3 TFs were identified in cultivars Hongbeibei (highly resistant), Zheza-301, Zhefen-702 (both resistant), Jinpeng-1, and Xianke-6 (both susceptible). Interaction network indicated that SlERF TFs could interact with mitogen-activated protein kinase (MAPK). Expression profiles of five ERF-B3 genes (, , , , and ) were detected by quantitative real-time-polymerase chain reaction (qRT-PCR) after TYLCV infection in five tomato cultivars. expression was upregulated in five tomato cultivars. The expressions of three genes (, , and ) were upregulated in Zheza-301 and Zhefen-702. and expressions were downregulated in Hongbeibei and Xianke-6, respectively. Yeast one-hybrid showed that the GCC-box binding ability of ERF-B3 TFs differed in resistant and susceptible tomato cultivars. Expression profiles were related to the GCC-box binding ability of SlERF TFs in resistant and susceptible tomato cultivars. The defense mechanism underlying the tomato's response to TYLCV involved a complicated network, which provided important information for us in breeding and genetic analysis.

Elevated Carbon Dioxide Altered Morphological and Anatomical Characteristics, Ascorbic Acid Accumulation, and Related Gene Expression during Taproot Development in Carrots.

The CO2 concentration in the atmosphere has increased significantly in recent decades and is projected to rise in the future. The effects of elevated CO2 concentrations on morphological and anatomical characteristics, and nutrient accumulation have been determined in several plant species. Carrot is an important vegetable and the effects of elevated CO2 on carrots remain unclear. To investigate the effects of elevated CO2 on the growth of carrots, two carrot cultivars ('Kurodagosun' and 'Deep purple') were treated with ambient CO2 (a[CO2], 400 µmol⋅mol-1) and elevated CO2 (e[CO2], 3000 µmol⋅mol-1) concentrations. Under e[CO2] conditions, taproot and shoot fresh weights and the root/shoot ratio of carrot significantly decreased as compared with the control group. Elevated CO2 resulted in obvious changes in anatomy and ascorbic acid accumulation in carrot roots. Moreover, the transcript profiles of 12 genes related to AsA biosynthesis and recycling were altered in response to e[CO2]. The 'Kurodagosun' and 'Deep purple' carrots differed in sensitivity to e[CO2]. The inhibited carrot taproot and shoot growth treated with e[CO2] could partly lead to changes in xylem development. This study provided novel insights into the effects of e[CO2] on the growth and development of carrots.

Morphological Characteristics, Anatomical Structure, and Gene Expression: Novel Insights into Cytokinin Accumulation during Carrot Growth and Development.

Cytokinins have been implicated in normal plant growth and development. These bioactive molecules are essential for cell production and expansion in higher plants. Carrot is an Apiaceae vegetable with great value and undergoes significant size changes over the process of plant growth. However, cytokinin accumulation and its potential roles in carrot growth have not been elucidated. To address this problem, carrot plants at five stages were collected, and morphological and anatomical characteristics and expression profiles of cytokinin-related genes were determined. During carrot growth and development, cytokinin levels were the highest at the second stage in the roots, whereas relatively stable levels were observed in the petioles and leaves. DcCYP735A2 showed high expression at stage 2 in the roots, which may contribute largely to the higher cytokinin level at this stage. However, expression of most metabolic genes did not follow a pattern similar to that of cytokinin accumulation, indicating that cytokinin biosynthesis was regulated through a complex network. Genes involved in cytokinin signal perception and transduction were also integrated to normal plant growth and development. The results from the present work suggested that cytokinins may regulate plant growth in a stage-dependent manner. Our work would shed novel insights into cytokinin accumulation and its potential roles during carrot growth. Further studies regarding carrot cytokinins may be achieved by modification of the genes involved in cytokinin biosynthesis, inactivation, and perception.