Plant Biotechnology Journal最新文献

Male sterility is an important agronomical trait in self-pollinating plants for producing cost-effective F1 hybrids to harness the heterosis. Still, large-scale development and maintenance of male sterile lines and restoring fertility in F1 hybrids pose significant challenges in plant hybrid breeding. Cotton is a self-pollinating crop and exhibits strong hybrid vigor. However, there are currently few breeding methods to achieve cost-effective production of F1 hybrid cotton. Here, we utilized novel functions of the Arabidopsis autophagy-related BECLIN1/ATG6 and a mutant of E3 ubiquitin ligase COP1 (COP1^L105A) genes in developing rescuable male sterility in cotton. We have generated multiple male-sterile (MS) and restorer (RS) cotton lines expressing BECLIN1 and COP1^L105A, respectively. Cytological observation showed that post-meiotic tapetal expression of BECLIN1 delays tapetum developmental programmed cell death (dPCD) by affecting reactive oxygen species (ROS) balance—this delay in dPCD results in early microspore defects and later small-sized flowers with indehiscent anthers. Furthermore, the evaluation of F1 hybrids developed by crossing MS and RS lines showed that early tapetal COP1^L105A expression abolishes expression of BECLIN1 resulting in normal tapetum degeneration, pollen development, and fertility. In addition, the F1 hybrid developed with MS and RS cotton lines in transgenic glass-house and net-house conditions showed the rescued fertility comparable with control plants (WT). In terms of cotton fiber productivity, the COP1^L105A-expressing transgenic cotton lines outperformed the WT. The current work effectively demonstrates the wider applicability of the new F1 cotton production system.

The functional stay-green trait is a major goal of rice breeding. Here, we cloned OsSCE1a encoding SUMO-conjugating enzyme from Yuanjiang common wild rice, which simultaneously regulates the functional stay-green trait and growth duration. Low expression or knocking out OsSCE1a corresponded to increased chlorophyll content, photosynthetic competence, N use efficiency and a shortened growth period without affecting yield. A natural MITE-transposon insertion/deletion in the OsSCE1a promoter is the functional variation that regulates these traits. OsSCE1a was selected during evolution and shows significant variation between indica and japonica rice. OsNAC2 interacts with the MITE to enhance OsSCE1a expression. Genetic manipulation of OsSCE1a revealed its potential for rice improvement. OsSCE1a-mediated SUMOylation of OsGS2 suppresses GS (involved in N assimilation) enzyme activity. OsSCE1a also regulates growth duration by SUMOylating the transcription factor such as OsGBP1, which regulates the expression of the key heading gene Ghd7. Our findings shed light on the role of SUMOylation in crops and provide a strategy for increasing agricultural productivity.

The protein crops known as lupins have been bred to accumulate low levels of antinutritional alkaloids, neglecting their potential as sources of valuable metabolites. Here, we engineered narrow-leafed lupin (NLL) to accumulate large amounts of a single alkaloid of industrial interest called (−)-sparteine. While (−)-sparteine is recognized as a key auxiliary molecule in chiral synthesis, its variable price and limited availability have prevented its large-scale use. We identified two enzymes that initiate the conversion of (−)-sparteine to a variety of alkaloids accumulating in NLL. The first one is a cytochrome P450 monooxygenase belonging to family 71 (CYP71D189), and the second one is a short-chain dehydrogenase/reductase (SDR1). We screened a non-GMO NLL mutant library and isolated a knockout in CYP71D189. The knockout displayed an altered metabolic profile where (−)-sparteine accounted for 96% of the alkaloid content in the seeds (GC–MS basis). The (−)-sparteine isolated from the mutant seeds was enantiomerically pure (99% enantiomeric excess). Apart from the altered alkaloid profile, the mutant did not have any noticeable phenotype. Our work demonstrates that (−)-sparteine is the precursor of most QAs in NLL and expands the current uses of NLL as a crop.

Prevention of severe COVID-19 disease by SARS-CoV-2 in high-risk patients, such as immuno-compromised individuals, can be achieved by administration of antibody prophylaxis, but producing antibodies can be costly. Plant expression platforms allow substantial lower production costs compared to traditional bio-manufacturing platforms depending on mammalian cells in bioreactors. In this study, we describe the expression, production and purification of the originally human COVA2-15 antibody in plants. Our plant-produced mAbs demonstrated comparable neutralizing activity with COVA2-15 produced in mammalian cells. Furthermore, they exhibited similar capacity to prevent SARS-CoV-2 infection in a hamster model. To further enhance these biosimilars, we performed three glyco- and protein engineering techniques. First, to increase antibody half-life, we introduced YTE-mutation in the Fc tail; second, optimization of N-linked glycosylation by the addition of a C-terminal ER-retention motif (HDEL), and finally; production of mAb in plant production lines lacking β-1,2-xylosyltransferase and α-1,3-fucosyltransferase activities (FX-KO). These engineered biosimilars exhibited optimized glycosylation, enhanced phagocytosis and NK cell activation capacity compared to conventional plant-produced S15 and M15 biosimilars, in some cases outperforming mammalian cell produced COVA2-15. These engineered antibodies hold great potential for enhancing in vivo efficacy of mAb treatment against COVID-19 and provide a platform for the development of antibodies against other emerging viruses in a cost-effective manner.

Resistant starch (RS) is a special kind of starch with beneficial effects on obesity, type 2 diabetes and other chronic complications. Breeding high-RS rice varieties is considered a valuable way to improve public health. However, most rice cultivars only contain an RS level lower than 2% in cooked rice, and cloning of RS genes is critical to improve RS levels in rice. The loss of function of Starch Synthases IIIa (SSIIIa) and SSIIIb, two amylopectin biosynthetic genes, could elevate RS levels up to 10%. Here, we performed a systematic genetic study of 14 amylopectin biosynthetic genes in the ssIIIa ssIIIb double mutant via genome editing, and investigated their effects on RS formation, the eating quality and grain yield. The results showed that deficiency in SSIIa, SSIVb or ISA2 under the ssIIIa ssIIIb background could each elevate RS content to above 14%, and the quadruple mutants of sbeI sbeIIb ssIIIa ssIIIb and sbeI ssIVb ssIIIa ssIIIb could further increase RS levels to over 18%. Furthermore, the eating quality of cooked rice and grain yield decreased along with the elevated RS contents, showing a trade-off among these traits. In these mutants, ssIIIa ssIIIb showed the balanced performance of RS and grain yield. This study provides insights into RS biosynthesis with a series of RS genes in the amylopectin biosynthesis pathway and practical strategy to breed high-RS rice varieties with balanced performance.

Very-long-chain (VLC) alkanes are major components of hydrophobic cuticular waxes that cover the aerial epidermis of land plants, serving as a waterproofing barrier to protect the plant against environmental stresses. The mechanism of VLC-alkane biosynthesis has been extensively elucidated in plants. However, little is known about the biosynthesis of long-chain alkanes (LC, C₁₃ ~ C₁₉) such as pentadecane in plants. Alkanes with different chain lengths are also major constituents of fossil fuels and thus the discovery of the alkane biosynthetic machinery in plants would provide a toolbox of enzymes for the production of renewable hydrocarbon sources and next generations of biofuels. The top leaves of Pogostemon cablin at young stage accumulate large amounts of LC-alkane pentadecane, making this plant an excellent system for the elucidation of LC-alkane biosynthetic machinery in plant. We show here that LC-alkane pentadecane biosynthesis in P. cablin involves an endoplasmic reticulum (ER)-localized complex made of PcCER1-LIKE3 and PcCER3, homologues of Arabidopsis ECERIFERUM1 (AtCER1) and AtCER3 proteins that are involved in Arabidopsis VLC-alkane biosynthesis. We reconstitute the biosynthesis of pentadecane in Nicotiana benthamiana by co-expression of PcCER1-LIKE3 and PcCER3 and further improve its production by silencing multifunctional acetyl-CoA carboxylases involved in fatty acid elongation pathway. Taken together, we uncovered the key biosynthetic machinery of LC-alkane pentadecane in P. cablin and demonstrated that using these newly identified enzymes to engineer this LC-alkane for liquid biofuel production in a heterologous plant host is possible.

As global temperatures rise, improving crop yields will require enhancing the thermotolerance of crops. One approach for improving thermotolerance is using bioengineering to increase the thermostability of enzymes catalysing essential biological processes. Photorespiration is an essential recycling process in plants that is integral to photosynthesis and crop growth. The enzymes of photorespiration are targets for enhancing plant thermotolerance as this pathway limits carbon fixation at elevated temperatures. We explored the effects of temperature on the activity of the photorespiratory enzyme glycerate kinase (GLYK) from various organisms and the homologue from the thermophilic alga Cyanidioschyzon merolae was more thermotolerant than those from mesophilic plants, including Arabidopsis thaliana. To understand enzyme features underlying the thermotolerance of C. merolae GLYK (CmGLYK), we performed molecular dynamics simulations using AlphaFold-predicted structures, which revealed greater movement of loop regions of mesophilic plant GLYKs at higher temperatures compared to CmGLYK. Based on these simulations, hybrid proteins were produced and analysed. These hybrid enzymes contained loop regions from CmGLYK replacing the most mobile corresponding loops of AtGLYK. Two of these hybrid enzymes had enhanced thermostability, with melting temperatures increased by 6 °C. One hybrid with three grafted loops maintained higher activity at elevated temperatures. Whilst this hybrid enzyme exhibited enhanced thermostability and a similar K_m for ATP compared to AtGLYK, its K_m for glycerate increased threefold. This study demonstrates that molecular dynamics simulation-guided structure-based recombination offers a promising strategy for enhancing the thermostability of other plant enzymes with possible application to increasing the thermotolerance of plants under warming climates.

The genetic improvement of rice eating and cooking quality (ECQ) is an important goal in rice breeding. It is important to understand the genetic regulation of ECQ at the genomic level for effective breeding to improve ECQ. However, the mechanisms underlying the improvement of ECQ of indica and japonica cultivars in southern China remain unclear. In this study, 290 rice cultivars (155 indica and 135 japonica cultivars) bred in southern China in the past 30 years were collected. Physicochemical indicators, namely, apparent amylose content (AAC), protein content (PC), lipid content and taste value, were measured and correlation analysis was performed. A decrease in AAC and PC was a crucial factor for the ECQ improvement of the rice cultivars in southern China. Genome-wide association analysis and selective domestication analysis preliminarily clarified that the comprehensive utilization of major and minor genes was an important genetic basis for improvement of ECQ. An elite allele, RAmy1A^A, with potential application in breeding to improve starch viscosity characteristics and ECQ, was mined. The Wx^b/OsmtSSB1L^T/OsDML4^G/RPBF^T/Du3^T and Wx^b/OsEro1^T/Glup3^G/OsNAC25^G/OsBEIIb^C/RAmy1A^A/FLO12^A gene modules, neither of which have been widely used, are proposed as the optimal allele combinations for ECQ improvement of indica and japonica cultivars in southern China. The results clarify the genetic regulation of rice ECQ improvement in southern China and provide novel genetic resources and breeding strategies for ECQ improvement in rice.

Crop diseases cause significant quality and yield losses to global crop products each year and are heavily controlled by chemicals along with very limited antibiotics composed of small molecules. However, these methods often result in environmental pollution and pest resistance, necessitating the development of new bio-controlling products to mitigate these hazards. To identify effective antimicrobial peptides (AMPs) considered as potential sources of future antibiotics, AMPs were screened from five bacterial strains showing antagonism against a representative phytopathogenic fungus (Rhizoctonia Solani) through the Bacillus subtilis expression system, which has been developed for identifying bacterial AMPs by displaying autolysis morphologies. A total of 5000 colonies were screened, and five displaying autolysis morphologies showed antagonism against R. solani. A novel AMP with the strongest antagonism efficiency was determined and tentatively named HR2-7, which is composed of 24 amino acids with an alpha-helical structure. HR2-7 has strong and broad-spectrum antimicrobial activity, tested against 10 g-positive and -negative bacteria and four phytopathogenic fungi by contact culture in plates with minimal lethal concentrations of 4.0 μM. When applied as purified peptide or in fermented B. subtilis culture solution, HR2-7 showed strong controlling efficiency on plants against diverse fungal and bacterial pathogens. Based on current understanding, HR2-7 is recognized as the first AMP derived from an agricultural antagonistic bacterium. It exhibits wide-ranging and notable antimicrobial efficacy, offering a supplementary approach for managing plant diseases, in addition to conventional chemical pesticides and antibiotics.