Genome-wide identification of 14-3-3 gene family and characterization of their expression in developmental stages of Solanum tuberosum under multiple biotic and abiotic stress conditions.

GF14 proteins are a family of conserved proteins involved in many cellular processes including transport, growth, metabolism, and stress response. However, only few reports are available regarding the 14-3-3 genes in potato. In this study, twelve 14-3-3 genes were detected in the potato genome. Based on their phylogenetic relationships, the StGF14 family members were categorized into two classes. Gene expression analysis demonstrated that StGF14h, StGF14a, and StGF14k had the highest gene expression, induced by abiotic and biotic stresses in all three tissues. The number of exons in 14-3-3 genes ranged from four to seven and most of these genes in the same subfamily had similar exon-intron patterns. The results of our study showed that the conserved motifs are similar in most of the proteins in each group. The intron-exon patterns and the composition of conserved motifs validated the 14-3-3 gene phylogenetic classification. According to the genome distribution results, 14-3-3 genes were located unevenly on the 12 Solanum tuberosum chromosomes. We find out 97 orthologous gene pairs between potato and Arabidopsis as well as 15 paralogous genes among potato genomes. Our results showed that GF-14 genes have an effective role in functional and molecular mechanisms in response to environmental stresses.

The 14-3-3 protein GF14c positively regulates immunity by modulating the protein homoeostasis of the GRAS protein OsSCL7 in rice.

Various types of transcription factors have been reported to be involved in plant-pathogen interactions by regulating defence-related genes. GRAS proteins, plant- specific transcription factors, have been shown to play essential roles in plant growth, development and stress responses. By performing a transcriptome study on rice early defence responses to Magnaporthe oryzae, we identified a GRAS protein, OsSCL7, which was induced by M. oryzae infection. We characterized the function of OsSCL7 in rice disease resistance. OsSCL7 was upregulated upon exposure to M. oryzae and pathogen-associated molecular pattern treatments, and knocking out OsSCL7 resulted in decreased disease resistance of rice to M. oryzae. In contrast, overexpression of OsSCL7 could improve rice disease resistance to M. oryzae. OsSCL7 was mainly localized in the nucleus and showed transcriptional activity. OsSCL7 can interact with GF14c, a 14-3-3 protein, and loss-of-function GF14c leads to enhanced susceptibility to M. oryzae. Additionally, OsSCL7 protein levels were reduced in the gf14c mutant and knocking out OsSCL7 affected the expression of a series of defence-related genes. Taken together, these findings uncover the important roles of OsSCL7 and GF14c in plant immunity and a potential mechanism by which plants fine-tune immunity by regulating the protein stability of a GRAS protein via a 14-3-3 protein.

Overexpression of OsGF14f Enhances Quantitative Leaf Blast and Bacterial Blight Resistance in Rice.

Although it is known that rice 14-3-3 family genes are involved in various defense responses, the functions of OsGF14f in response to diseases have not been reported. Here, we showed that the transcription of OsGF14f was significantly induced by leaf blast infection, and the overexpression of OsGF14f quantitatively enhanced resistance to leaf blast and bacterial blight in rice. Further analysis showed that the expression levels of salicylic acid (SA) pathway-associated genes (PAL1, NH1, PR1a and PR10) in the OsGF14f-overexpressing plants, were higher than those in wild-type plants after inoculation with the blast isolate (Magnaporthe oryzae Barr). In addition, the expression level of OsGF14f was significantly induced after SA treatment, and higher endogenous SA levels were observed in the OsGF14f-overexpressing plants compared with that in wild-type plants, especially after blast challenge. Taken together, these results suggest that OsGF14f positively regulates leaf blast and bacterial blight resistance in rice via the SA-dependent signaling pathway.

The 14-3-3 protein GF14f negatively affects grain filling of inferior spikelets of rice (Oryza sativa L.).

In rice (Oryza sativa L.), later flowering inferior spikelets (IS), which are located on proximal secondary branches, fill slowly and produce smaller and lighter grains than earlier flowering superior spikelets (SS). Many genes have been reported to be involved in poor grain filling of IS, however the underlying molecular mechanisms remain unclear. The present study determined that GF14f, a member of the 14-3-3 protein family, showed temporal and spatial differences in expression patterns between SS and IS. Using GF14f-RNAi plants, we observed that a reduction in GF14f expression in the endosperm resulted in a significant increase in both grain length and weight, which in turn improved grain yield. Furthermore, pull-down assays indicated that GF14f interacts with enzymes that are involved in sucrose breakdown, starch synthesis, tricarboxylic acid (TCA) cycle and glycolysis. At the same time, an increase in the activity of sucrose synthase (SuSase), adenosine diphosphate-glucose pyrophosphorylase (AGPase), and starch synthase (StSase) was observed in the GF14f-RNAi grains. Comprehensive analysis of the proteome and metabolite profiling revealed that the abundance of proteins related to the TCA cycle, and glycolysis increased in the GF14f-RNAi grains together with several carbohydrate intermediates. These results suggested that GF14f negatively affected grain development and filling, and the observed higher abundance of the GF14f protein in IS compared with SS may be responsible for poor IS grain filling. The study provides insights into the molecular mechanisms underlying poor grain filling of IS and suggests that GF14f could serve as a potential tool for improving rice grain filling.

OsGF14e positively regulates panicle blast resistance in rice.

Though GF14e has been reported to negatively regulate bacterial blight and sheath blight resistance in rice, its effect on panicle blast, the most destructive disease in rice is still unknown. In the present study, we identified that GF14e was highly expressed in panicles and was induced in panicles infected by blast pathogen. Overexpression of GF14e enhances resistance to panicle blast whereas silencing GF14e results in increased susceptibility to panicle blast, suggesting that GF14e plays a positive role in quantitative panicle blast resistance in rice. Our results also demonstrate that GF14e is regulated by WRKY71 and GF14e-mediated panicle blast resistance is related to activation of SA-dependent pathway and suppression of JA-dependent pathway. The functional confirmation of GF14e in panicle blast resistance makes it to be a promising target in molecular rice breeding.

Comparative Proteomic Analysis Provides New Insights into Improved Grain-filling in Ratoon Season Rice.

Grain-filling of rice spikelets (particularly for the later flowering inferior spikelets) is an important characteristic that affects both quality and yield. Rice ratooning technology is used to cultivate a second crop from dormant buds that sprout from stubble left after the first harvest. This study used two rice varieties, the conventional indica rice 'Jinhui 809' and the hybrid indica-japonica rice 'Yongyou 1540', to assess the impact of rice ratooning on grain-filling. The results indicated that the grain-filling process in inferior spikelets of ratoon season rice (ISR) showed significant improvement compared to inferior spikelets of main crop (late season) rice (ISL). This improvement was evident in the earlier onset of rapid grain-filling, higher seed-setting percentage, and improved grain quality. A label-free quantitative proteomic analysis using mass spectrometry identified 1724 proteins with significant abundance changes, shedding light on the molecular mechanisms behind the improved grain-filling in ISR. The functional analysis of these proteins indicated that ratooning stimulated the metabolic processes of sucrose-starch, trehalose, and hormones in rice inferior spikelets, leading to enhanced enzyme activities related to starch synthesis, elevated concentrations of trehalose-6-phosphate (T6P), indole-3-acetic acid (IAA) and zeatin riboside (ZR) during the active grain-filling phase. This research highlighted the importance of the GF14f protein as a key regulator in the grain-filling process of ISR. It revealed that GF14f transcriptional and protein levels declined more rapidly in ISR compared to ISL during grain-filling. Additionally, the GF14f-RNAi plants specific to the endosperm exhibited improved quality in inferior spikelets. These findings suggest that the enhancement of starch synthesis, increased levels of IAA, ZR, and T6P, along with the rapid decrease in GF14f protein, play a role in enhancing grain-filling in ratoon season rice.

GF14f gene is negatively associated with yield and grain chalkiness under rice ratooning.

Background: Ratoon rice cropping has been shown to provide new insights into overcoming the current challenges of rice production in southern China. However, the potential mechanisms impacting yield and grain quality under rice ratooning remain unclear. Methods: In this study, changes in yield performance and distinct improvements in grain chalkiness in ratoon rice were thoroughly investigated, using physiological, molecular and transcriptomic analysis. Results: Rice ratooning induced an extensive carbon reserve remobilization in combination with an impact on grain filling, starch biosynthesis, and ultimately, an optimization in starch composition and structure in the endosperm. Furthermore, these variations were shown to be associated with a protein-coding gene: GF14f (encoding GF14f isoform of 14-3-3 proteins) and such gene negatively impacts oxidative and environmental resistance in ratoon rice. Conclusion: Our findings suggested that this genetic regulation by GF14f gene was the main cause leading to changes in rice yield and grain chalkiness improvement of ratoon rice, irrespective of seasonal or environmental effects. A further significance was to see how yield performance and grain quality of ratoon rice were able to be achieved at higher levels via suppression of GF14f.

Genome-wide analysis of the G-box regulating factors protein family reveals its roles in response to Sclerotinia sclerotiorum infection in rapeseed (Brassica napus L.).

The G-box regulating factors (GRFs) are involved in a wide array of signal transduction pathway and play important roles in plant physiological and developmental processes and stress responses. The GRF proteins have previously been described in several plant species, but not in rapeseed (Brassica napus L.). In this study, we carried out genome-wide analysis of GRFs in B. napus based on the available genome sequence information, and analyzed their expression in different tissues under different hormone treatments and after inoculation with Sclerotinia sclerotiorum. We identified 46 putative BnaGRF genes in rapeseed, unevenly distributed on 18 chromosomes. Like the 14-3-3 proteins in other plant species, the 46 putative BnaGRFs could be classified into two major evolutionary branches: epsilon (ε) group and non-epsilon (non-ε) group. Evolutionary analysis indicated that the BnaGRF gene family expanded in both groups much before speciation. We discovered an expansion of the 14-3-3 gene family that likely occurred during a recent gene duplication event. Collinearity analysis revealed that most of the BnaGRF genes shared syntenic relationships. Global gene expression profiling of BnaGRFs by RNA-seq analysis showed 41.3% (19/46) response to S. sclerotiorum infection, and this response was probably mediated through jasmonic acid (JA) and salicylic acid (SA) signaling pathways. These results provide key insights into the role of 14-3-3s in the biotic stress response and enhance our understanding of their multiple functions in B. napus.