The Barley HvSTP13GR mutant triggers resistance against biotrophic fungi

High-yielding and stress resistant crops are essential to ensure future food supply. Barley is an important crop to feed livestock and to produce malt, but the annual yield is threatened by pathogen infections. Pathogens can trigger an altered sugar partitioning in the host plant, that possibly leads to an advantage for the pathogen. Hampering these processes represents a promising strategy to potentially increase resistance. We analyzed the response of the barley monosaccharide transporter HvSTP13 towards biotic stress and its potential use for plant protection. The expression of HvSTP13 increased upon bacterial and fungal PAMP application, suggesting a PAMP-triggered signaling that converged on the transcriptional induction of the gene. Promoter studies indicate a region that is likely targeted by transcription factors downstream of PAMP-triggered immunity pathways. We confirmed that the non-functional HvSTP13GR variant confers resistance against an economically relevant biotrophic rust fungus, in barley. In addition, we established targeted CRISPR/Cas9 cytosine base editing in barley protoplasts to generate alternative HvSTP13 mutants and characterized the sugar transport activity and subcellular localization of the proteins. These mutants represent promising variants for future resistance analysis. Our experimental setup provides basal prerequisites to further decode the role of HvSTP13 in response to biological stress. Moreover, in line with other studies, our experiments indicate that the alteration of sugar partitioning pathways, in a host pathogen interaction, is a promising approach to achieve broad and durable resistance in plants.

Mobilizing Vacuolar Sugar Increases Vegetative Triacylglycerol Accumulation

Photosynthetically derived sugars provide carbon skeletons for metabolism and carbon signals that favor anabolism. The amount of sugar available for fatty acid (FA) and triacylglycerol (TAG) synthesis depends on sugar compartmentation, transport, and demands from competing pathways. We are exploring the influence of sugar partitioning between the vacuole and cytoplasm on FA synthesis in Arabidopsis by building on our previous finding that reduced leaf sugar export in the sucrose-proton symporter2 (suc2) mutant, in combination with impaired starch synthesis in the ADP-glucose pyrophosphorylase (adg1) mutant, accumulates higher sugar levels and increased total FA and TAG compared to the wild type parent. Here we sought to relocalize sugar from the vacuole to the cytoplasm to drive additional FA/TAG synthesis and growth. Arabidopsis suc2 adg1 was therefore crossed with tonoplast monosaccharide transporter mutants tmt1 and tmt2 and overexpression of the sucrose/proton cotransporter SUC4 in which tmt1 tmt2 impairs sugar transport to the vacuole from the cytoplasm and SUC4 overexpression enhances sugar transport in the reverse direction from the vacuole to the cytoplasm. A resulting homozygous suc2 adg1 tmt1 tmt2 SUC4 line was used to test the hypothesis that increased intracellular carbon supply in the form of sugars would increase both FA and TAG accumulation. The data shows that relative to suc2 adg1, suc2 adg1 tmt1 tmt2 SUC4 significantly increases leaf total FA content by 1.29-fold to 10.9% of dry weight and TAG by 2.4-fold to 2.88%, supporting the hypothesis that mobilizing vacuolar sugar is a valid strategy for increasing vegetative oil accumulation.

Optimizing the CRISPR/Cas9 system for genome editing in grape by using grape promoters

The efficacy of the CRISPR/Cas9 system in grapevine (Vitis vinifera L.) has been documented, but the optimization of this system, as well as CRISPR/Cas9-mediated multiplex genome editing, has not been explored in this species. Herein, we identified four VvU3 and VvU6 promoters and two ubiquitin (UBQ) promoters in grapevine and demonstrated that the use of the identified VvU3/U6 and UBQ2 promoters could significantly increase the editing efficiency in grape by improving the expression of sgRNA and Cas9, respectively. Furthermore, we conducted multiplex genome editing using the optimized CRISPR/Cas9 vector that contained the conventional multiple sgRNA expression cassettes or the polycistronic tRNA-sgRNA cassette (PTG) by targeting the sugar-related tonoplastic monosaccharide transporter (TMT) family members TMT1 and TMT2, and the overall editing efficiencies were higher than 10%. The simultaneous editing of TMT1 and TMT2 resulted in reduced sugar levels, which indicated the role of these two genes in sugar accumulation in grapes. Moreover, the activities of the VvU3, VvU6, and UBQ2 promoters in tobacco genome editing were demonstrated by editing the phytoene desaturase (PDS) gene in Nicotiana benthamiana leaves. Our study provides materials for the optimization of the CRISPR/Cas9 system. To our knowledge, our simultaneous editing of the grape TMT family genes TMT1 and TMT2 constitutes the first example of multiplex genome editing in grape. The multiplex editing systems described in this manuscript expand the toolbox of grape genome editing, which would facilitate basic research and molecular breeding in grapevine.

Comprehensive Analysis of Evolutionary Characterization and Expression for Monosaccharide Transporter Family Genes in Nelumbo nucifera

Sugar transporters, an important class of transporters for sugar function, regulate many processes associated with growth, maturation, and senescence processes in plants. In this study, a total of 35 NuMSTs were identified in the Nelumbo nucifera genome and grouped by conserved domains and phylogenetic analysis. Additionally, we identified 316 MST genes in 10 other representative plants and performed a comparative analysis with Nelumbo nucifera genes, including evolutionary trajectory, gene duplication, and expression pattern. A large number of analyses across plants and algae indicated that the MST family could have originated from STP and Glct, expanding to form STP and SFP by dispersed duplication. Finally, a quantitative real-time polymerase chain reaction and cis-element analysis showed that some of them may be regulated by plant hormones (e.g., abscisic acid), biotic stress factors, and abiotic factors (e.g., drought, excessive cold, and light). We found that under the four abiotic stress conditions, only NuSTP5 expression was upregulated, generating a stress response, and ARBE and LTR were present in NuSTP5. In summary, our findings are significant for understanding and exploring the molecular evolution and mechanisms of NuMSTs in plants.

A Heat Stress Responsive NAC Transcription Factor Heterodimer Plays Key Roles in Rice Grain Filling

High temperature often leads to the failure of grain filling in rice (Oryza sativa) to cause yield loss, while the mechanism is not well elucidated yet. Here, we report that two seed-specific NAM/ATAF/CUC domain transcription factors, ONAC127 and ONAC129, are responsive to heat stress and involved in the grain filling process of rice. ONAC127 and ONAC129 are dominantly expressed in the pericarp and can form a heterodimer during rice grain filling. CRISPR/Cas9 induced mutants and overexpression lines were then generated to investigate the functions of these two transcription factors. Interestingly, both knock-out and overexpression plants showed incomplete grain filling and shrunken grains, which became more severe under heat stress. Transcriptome analysis revealed that ONAC127 and ONAC129 mainly regulate stimulus response and nutrient transport. ChIP-seq analysis identified that the direct targets of ONAC127 and ONAC129 in developing rice seeds include monosaccharide transporter OsMST6, sugar transporter OsSWEET4, calmodulin-like protein OsMSR2 and AP2/ERF factor OsEATB. These results suggest that ONAC127 and ONAC129 may regulate grain filling through affecting sugar transportation and abiotic stress responses. Overall, this study demonstrates a transcriptional regulatory network involving ONAC127 and ONAC129 and coordinating multiple pathways to modulate seed development and heat stress response at rice reproductive stage.

Genome-Wide Identification and Expression Profiling of Monosaccharide Transporter Genes Associated with High Harvest Index Values in Rapeseed (Brassica napus L.)

Sugars are important throughout a plant’s lifecycle. Monosaccharide transporters (MST) are essential sugar transporters that have been identified in many plants, but little is known about the evolution or functions of MST genes in rapeseed (Brassica napus). In this study, we identified 175 MST genes in B. napus, 87 in Brassica oleracea, and 83 in Brassica rapa. These genes were separated into the sugar transport protein (STP), polyol transporter (PLT), vacuolar glucose transporter (VGT), tonoplast monosaccharide transporter (TMT), inositol transporter (INT), plastidic glucose transporter (pGlcT), and ERD6-like subfamilies, respectively. Phylogenetic and syntenic analysis indicated that gene redundancy and gene elimination have commonly occurred in Brassica species during polyploidization. Changes in exon-intron structures during evolution likely resulted in the differences in coding regions, expression patterns, and functions seen among BnMST genes. In total, 31 differentially expressed genes (DEGs) were identified through RNA-seq among materials with high and low harvest index (HI) values, which were divided into two categories based on the qRT-PCR results, expressed more highly in source or sink organs. We finally identified four genes, including BnSTP5, BnSTP13, BnPLT5, and BnERD6-like14, which might be involved in monosaccharide uptake or unloading and further affect the HI of rapeseed. These findings provide fundamental information about MST genes in Brassica and reveal the importance of BnMST genes to high HI in B. napus.

Molecular and physiological characterization of the monosaccharide transporters gene family in Medicago truncatula

Monosaccharide transporters (MSTs) represent key components of the carbon transport and partitioning mechanisms in plants, mediating the cell-to-cell and long-distance distribution of a wide variety of monosaccharides. In this study, we performed a thorough structural, molecular, and physiological characterization of the monosaccharide transporter gene family in the model legume Medicago truncatula. The complete set of MST family members was identified with a novel bioinformatic approach. Prolonged darkness was used as a test condition to identify the relevant transcriptomic and metabolic responses combining MST transcript profiling and metabolomic analysis. Our results suggest that MSTs play a pivotal role in the efficient partitioning and utilization of sugars, and possibly in the mechanisms of carbon remobilization in nodules upon photosynthate-limiting conditions, as nodules are forced to acquire a new role as a source of both C and N.