Aspartate Residues in a Forisome-Forming SEO Protein Are Critical for Protein Body Assembly and Ca2+ Responsiveness.

Forisomes are protein bodies known exclusively from sieve elements of legumes. Forisomes contribute to the regulation of phloem transport due to their unique Ca2+-controlled, reversible swelling. The assembly of forisomes from sieve element occlusion (SEO) protein monomers in developing sieve elements and the mechanism(s) of Ca2+-dependent forisome contractility are poorly understood because the amino acid sequences of SEO proteins lack conventional protein-protein interaction and Ca2+-binding motifs. We selected amino acids potentially responsible for forisome-specific functions by analyzing SEO protein sequences in comparison to those of the widely distributed SEO-related (SEOR), or SEOR proteins. SEOR proteins resemble SEO proteins closely but lack any Ca2+ responsiveness. We exchanged identified candidate residues by directed mutagenesis of the Medicago truncatula SEO1 gene, expressed the mutated genes in yeast (Saccharomyces cerevisiae) and studied the structural and functional phenotypes of the forisome-like bodies that formed in the transgenic cells. We identified three aspartate residues critical for Ca2+ responsiveness and two more that were required for forisome-like bodies to assemble. The phenotypes observed further suggested that Ca2+-controlled and pH-inducible swelling effects in forisome-like bodies proceeded by different yet interacting mechanisms. Finally, we observed a previously unknown surface striation in native forisomes and in recombinant forisome-like bodies that could serve as an indicator of successful forisome assembly. To conclude, this study defines a promising path to the elucidation of the so-far elusive molecular mechanisms of forisome assembly and Ca2+-dependent contractility.

eIFiso4G Augments the Synthesis of Specific Plant Proteins Involved in Normal Chloroplast Function.

The plant-specific translation initiation complex eIFiso4F is encoded by three genes in Arabidopsis (Arabidopsis thaliana)-genes encoding the cap binding protein eIFiso4E (eifiso4e) and two isoforms of the large subunit scaffolding protein eIFiso4G (i4g1 and i4g2). To quantitate phenotypic changes, a phenomics platform was used to grow wild-type and mutant plants (i4g1, i4g2, i4e, i4g1 x i4g2, and i4g1 x i4g2 x i4e [i4f]) under various light conditions. Mutants lacking both eIFiso4G isoforms showed the most obvious phenotypic differences from the wild type. Two-dimensional differential gel electrophoresis and mass spectrometry were used to identify changes in protein levels in plants lacking eIFiso4G. Four of the proteins identified as measurably decreased and validated by immunoblot analysis were two light harvesting complex binding proteins 1 and 3, Rubisco activase, and carbonic anhydrase. The observed decreased levels for these proteins were not the direct result of decreased transcription or protein instability. Chlorophyll fluorescence induction experiments indicated altered quinone reduction kinetics for the double and triple mutant plants with significant differences observed for absorbance, trapping, and electron transport. Transmission electron microscopy analysis of the chloroplasts in mutant plants showed impaired grana stacking and increased accumulation of starch granules consistent with some chloroplast proteins being decreased. Rescue of the i4g1 x i4g2 plant growth phenotype and increased expression of the validated proteins to wild-type levels was obtained by overexpression of eIFiso4G1. These data suggest a direct and specialized role for eIFiso4G in the synthesis of a subset of plant proteins.

Characterization of Brassica rapa RAP2.4-Related Proteins in Stress Response and as CUL3-Dependent E3 Ligase Substrates.

The turnip Brassica rapa has important economic value and represents a good model system to study gene function in crop plants. ERF/AP2 transcription factors are a major group of proteins that are often involved in regulating stress-responses and developmental programs. Some ERF/AP2 proteins are targets of CULLIN3-based E3 ligases that use BTB/POZ-MATH proteins as substrate receptors. These receptors bind the transcription factor and facilitate their ubiquitylation and subsequent degradation via the 26S proteasome. Here, we show tissue and stress-dependent expression patterns for three Brassica rapa ERF/AP2 proteins that are closely related to Arabidopsis thaliana AtRAP2.4. Cloning of the Brassica genes showed that the corresponding proteins can assemble with a BPM protein and CULLIN3, and that they are instable in a 26S proteasome dependent manner. This work demonstrates the conserved nature of the ERF/AP2-CULLIN3-based E3 ligase interplay, and represents a first step to analyze their function in a commercially relevant crop plant.

Effect of low-temperature storage on the content of folate, vitamin B6 , ascorbic acid, chlorogenic acid, tyrosine, and phenylalanine in potatoes.

BACKGROUND: Changes in the metabolite composition of potato tubers during low-temperature storage can affect their nutritional value, susceptibility to bruising, and processing qualities. Here, we measured changes in the amounts of folate, vitamin B6 , and vitamin C, and the blackspot pigment precursors chlorogenic acid and tyrosine, as well as phenylalanine, in five potato varieties stored at 7.8 °C for 8 months in 2015 and 2016. RESULTS: Folate content increased in all varieties in both years during low-temperature storage, with statistically significant changes occurring in six out of eight conditions. Increase rates ranged from 11% to 141%. Vitamin B6 content increased in all varieties during the storage period, but changes were statistically significant in only two out of eight conditions. Increase rates ranged from 5% to 24%. Ascorbic acid content decreased in all varieties in both years during the storage period. Decrease rates ranged from 16% to 78%, and were statistically significant in seven out of eight conditions. For chlorogenic acid, no consistent trend was observed. Changes varied between -14% and +14%, but none was statistically significant. Tyrosine content increased in all varieties in both years, except in Sage Russet in 2015. Increase rates ranged from 19% to 238% and were statistically significant in three out of seven conditions. Changes in phenylalanine content were very similar to those observed for tyrosine, with increases up to 272% in Teton Russet. CONCLUSIONS: These results show that storage at low temperature substantially affects tuber nutritional quality and biochemical bruising potential. © 2019 Society of Chemical Industry.

Meeting report: processing, translation, decay - three ways to keep RNA sizzling.

This meeting report highlights key trends that emerged from a conference entitled Post-Transcriptional Gene Regulation in Plants, which was held 14-15 July 2016, as a satellite meeting of the annual meeting of the American Society of Plant Biologists in Austin, Texas. The molecular biology of RNA is emerging as an integral part of the framework for plants' responses to environmental challenges such as drought and heat, hypoxia, nutrient deprivation, light and pathogens. Moreover, the conference illustrated how a multitude of customized and pioneering omics-related technologies are being applied, more and more often in combination, to describe and dissect the complexities of gene expression at the post-transcriptional level.

Arabidopsis thaliana PDX1.2 is critical for embryo development and heat shock tolerance.

MAIN CONCLUSION: PDX1.2 is expressed in the basal part of the globular-stage embryo, and plays critical roles in development, hypocotyl elongation, and stress response. The Arabidopsis thaliana PDX1.2 protein belongs to a small family of three members. While PDX1.1 and PDX1.3 have been extensively described and are well established to function in vitamin B6 biosynthesis, the biological role of PDX1.2 still remains elusive. Here, we show that PDX1.2 is expressed early in embryo development, and that heat shock treatment causes a strong up-regulation of the gene. Using a combined genetic approach of T-DNA insertion lines and expression of artificial micro RNAs, we can show that PDX1.2 is critically required for embryo development, and for normal hypocotyl elongation. Plants with reduced PDX1.2 expression also display reduced primary root growth after heat shock treatments. The work overall provides a set of important new findings that give greater insights into the developmental role of PDX1.2 in plants.

The Arabidopsis CstF64-Like RSR1/ESP1 Protein Participates in Glucose Signaling and Flowering Time Control.

Mechanisms for sensing and regulating metabolic processes at the cellular level are critical for the general physiology and development of living organisms. In higher plants, sugar signaling is crucial for adequate regulation of carbon and energy metabolism and affects virtually every aspect of development. Although many genes are regulated by sugar levels, little is known on how sugar levels are measured by plants. Several components of the sugar signaling network have been unraveled and demonstrated to have extensive overlap with hormone signaling networks. Here we describe the reduced sugar response1-1 (rsr1-1) mutant as a new early flowering mutant that displays decreased sensitivity to abscisic acid. Both hexokinase1 (HXK1)-dependent and glucose phosphorylation-independent signaling is reduced in rsr1-1. Map-based identification of the affected locus demonstrated that rsr1-1 carries a premature stop codon in the gene for a CstF64-like putative RNA processing factor, ESP1, which is involved in mRNA 3'-end formation. The identification of RSR1/ESP1 as a nuclear protein with a potential threonine phosphorylation site may explain the impact of protein phosphorylation cascades on sugar-dependent signal transduction. Additionally, RSR1/ESP1 may be a crucial factor in linking sugar signaling to the control of flowering time.