Impaired KIN10 function restores developmental defects in the Arabidopsis trehalose 6-phosphate synthase1 (tps1) mutant.

Sensing carbohydrate availability is essential for plants to coordinate their growth and development. In Arabidopsis thaliana, TREHALOSE 6-PHOSPHATE SYNTHASE 1 (TPS1) and its product, trehalose 6-phosphate (T6P), are important for the metabolic control of development. tps1 mutants are embryo-lethal and unable to flower when embryogenesis is rescued. T6P regulates development in part through inhibition of SUCROSE NON-FERMENTING1 RELATED KINASE1 (SnRK1). Here, we explored the role of SnRK1 in T6P-mediated plant growth and development using a combination of a mutant suppressor screen and genetic, cellular and transcriptomic approaches. We report nonsynonymous amino acid substitutions in the catalytic KIN10 and regulatory SNF4 subunits of SnRK1 that can restore both embryogenesis and flowering of tps1 mutant plants. The identified SNF4 point mutations disrupt the interaction with the catalytic subunit KIN10. Contrary to the common view that the two A. thaliana SnRK1 catalytic subunits act redundantly, we found that loss-of-function mutations in KIN11 are unable to restore embryogenesis and flowering, highlighting the important role of KIN10 in T6P signalling.

Removing auto-activators from yeast-two-hybrid assays by conditional negative selection.

Yeast-two-hybrid (Y2H) is widely used as a strategy to detect protein-protein interactions (PPIs). Recent advancements have made it possible to generate and analyse genome-wide PPI networks en masse by coupling Y2H with next-generation sequencing technology. However, one of the major challenges of yeast two-hybrid assay is the large amount of false-positive hits caused by auto-activators (AAs), which are proteins that activate the reporter genes without the presence of an interacting protein partner. Here, we have developed a negative selection to minimize these auto-activators by integrating the pGAL2-URA3 fragment into the yeast genome. Upon activation of the pGAL2 promoter by an AA, yeast cells expressing URA3 cannot grow in media supplemented with 5-Fluoroorotic acid (5-FOA). Hence, we selectively inhibit the growth of yeast cells expressing auto-activators and thus minimizing the amount of false-positive hits. Here, we have demonstrated that auto-activators can be successfully removed from a Marchantia polymorpha cDNA library using pGAL2-URA3 and 5-FOA treatment, in liquid and solid-grown cultures. Furthermore, since URA3 can also serve as a marker for uracil autotrophy, we propose that our approach is a valuable addition to any large-scale Y2H screen.

AtRsgA from Arabidopsis thaliana is important for maturation of the small subunit of the chloroplast ribosome.

Plastid ribosomes are very similar in structure and function to the ribosomes of their bacterial ancestors. Since ribosome biogenesis is not thermodynamically favorable under biological conditions it requires the activity of many assembly factors. Here we have characterized a homolog of bacterial RsgA in Arabidopsis thaliana and show that it can complement the bacterial homolog. Functional characterization of a strong mutant in Arabidopsis revealed that the protein is essential for plant viability, while a weak mutant produced dwarf, chlorotic plants that incorporated immature pre-16S ribosomal RNA into translating ribosomes. Physiological analysis of the mutant plants revealed smaller, but more numerous, chloroplasts in the mesophyll cells, reduction of chlorophyll a and b, depletion of proplastids from the rib meristem and decreased photosynthetic electron transport rate and efficiency. Comparative RNA sequencing and proteomic analysis of the weak mutant and wild-type plants revealed that various biotic stress-related, transcriptional regulation and post-transcriptional modification pathways were repressed in the mutant. Intriguingly, while nuclear- and chloroplast-encoded photosynthesis-related proteins were less abundant in the mutant, the corresponding transcripts were increased, suggesting an elaborate compensatory mechanism, potentially via differentially active retrograde signaling pathways. To conclude, this study reveals a chloroplast ribosome assembly factor and outlines the transcriptomic and proteomic responses of the compensatory mechanism activated during decreased chloroplast function.

Elucidating gene function and function evolution through comparison of co-expression networks of plants.

The analysis of gene expression data has shown that transcriptionally coordinated (co-expressed) genes are often functionally related, enabling scientists to use expression data in gene function prediction. This Focused Review discusses our original paper (Large-scale co-expression approach to dissect secondary cell wall formation across plant species, Frontiers in Plant Science 2:23). In this paper we applied cross-species analysis to co-expression networks of genes involved in cellulose biosynthesis. We showed that the co-expression networks from different species are highly similar, indicating that whole biological pathways are conserved across species. This finding has two important implications. First, the analysis can transfer gene function annotation from well-studied plants, such as Arabidopsis, to other, uncharacterized plant species. As the analysis finds genes that have similar sequence and similar expression pattern across different organisms, functionally equivalent genes can be identified. Second, since co-expression analyses are often noisy, a comparative analysis should have higher performance, as parts of co-expression networks that are conserved are more likely to be functionally relevant. In this Focused Review, we outline the comparative analysis done in the original paper and comment on the recent advances and approaches that allow comparative analyses of co-function networks. We hypothesize that in comparison to simple co-expression analysis, comparative analysis would yield more accurate gene function predictions. Finally, by combining comparative analysis with genomic information of green plants, we propose a possible composition of cellulose biosynthesis machinery during earlier stages of plant evolution.

Identification of nutrient-responsive Arabidopsis and rapeseed microRNAs by comprehensive real-time polymerase chain reaction profiling and small RNA sequencing.

Comprehensive expression profiles of Arabidopsis (Arabidopsis thaliana) MIRNA genes and mature microRNAs (miRs) are currently not available. We established a quantitative real-time polymerase chain reaction platform that allows rapid and sensitive quantification of 177 Arabidopsis primary miR transcripts (pri-miRs). The platform was used to detect phosphorus (P) or nitrogen (N) status-responsive pri-miR species. Several pri-miR169 species as well as pri-miR398a were found to be repressed during N limitation, whereas during P limitation, pri-miR778, pri-miR827, and pri-miR399 species were induced and pri-miR398a was repressed. The corresponding responses of the biologically active, mature miRs were confirmed using specific stem-loop reverse transcription primer quantitative polymerase chain reaction assays and small RNA sequencing. Interestingly, the latter approach also revealed high abundance of some miR star strands. Bioinformatic analysis of small RNA sequences with a modified miRDeep algorithm led to the identification of the novel P limitation-induced miR2111, which is encoded by two loci in the Arabidopsis genome. Furthermore, miR2111, miR169, a miR827-like sequence, and the abundances of several miR star strands were found to be strongly dependent on P or N status in rapeseed (Brassica napus) phloem sap, flagging them as candidate systemic signals. Taken together, these results reveal the existence of complex small RNA-based regulatory networks mediating plant adaptation to mineral nutrient availability.