Plant Proteomic Data Acquisition and Data Analyses: Lessons from Spaceflight.

Proteomics has the capacity to identify and quantify the proteins present in a sample. The technique has been used extensively across all model organisms to study various physiological processes and signaling pathways. In addition to providing a global view of regulatory processes inside a cell, proteomics can also be used to identify candidate genes and retrieve information on alternative isoforms of known proteins. Here, we provide protocols for protein extraction from Arabidopsis thaliana seedlings and describe analysis techniques used after data collection. This approach was originally used for the Biological Research in Canisters (BRIC) 20 spaceflight experiment but is valid for any ground-based or flight experiment. Extraction protocols for soluble and membrane proteins and basic analysis and quality metrics for MS/MS data are provided. Avenues for data analysis post-MS/MS data acquisition and details of software that can be used in gathering structural data on proteins of interest are also included. Use of differential abundance and network-based approaches for proteomics data analyses can reveal regulatory patterns not apparent through differential abundance or transcriptomic data alone.

Spaceflight induces novel regulatory responses in Arabidopsis seedling as revealed by combined proteomic and transcriptomic analyses.

BACKGROUND: Understanding of gravity sensing and response is critical to long-term human habitation in space and can provide new advantages for terrestrial agriculture. To this end, the altered gene expression profile induced by microgravity has been repeatedly queried by microarray and RNA-seq experiments to understand gravitropism. However, the quantification of altered protein abundance in space has been minimally investigated. RESULTS: Proteomic (iTRAQ-labelled LC-MS/MS) and transcriptomic (RNA-seq) analyses simultaneously quantified protein and transcript differential expression of three-day old, etiolated Arabidopsis thaliana seedlings grown aboard the International Space Station along with their ground control counterparts. Protein extracts were fractionated to isolate soluble and membrane proteins and analyzed to detect differentially phosphorylated peptides. In total, 968 RNAs, 107 soluble proteins, and 103 membrane proteins were identified as differentially expressed. In addition, the proteomic analyses identified 16 differential phosphorylation events. Proteomic data delivered novel insights and simultaneously provided new context to previously made observations of gene expression in microgravity. There is a sweeping shift in post-transcriptional mechanisms of gene regulation including RNA-decapping protein DCP5, the splicing factors GRP7 and GRP8, and AGO4,. These data also indicate AHA2 and FERONIA as well as CESA1 and SHOU4 as central to the cell wall adaptations seen in spaceflight. Patterns of tubulin-α 1, 3,4 and 6 phosphorylation further reveal an interaction of microtubule and redox homeostasis that mirrors osmotic response signaling elements. The absence of gravity also results in a seemingly wasteful dysregulation of plastid gene transcription. CONCLUSIONS: The datasets gathered from Arabidopsis seedlings exposed to microgravity revealed marked impacts on post-transcriptional regulation, cell wall synthesis, redox/microtubule dynamics, and plastid gene transcription. The impact of post-transcriptional regulatory alterations represents an unstudied element of the plant microgravity response with the potential to significantly impact plant growth efficiency and beyond. What's more, addressing the effects of microgravity on AHA2, CESA1, and alpha tubulins has the potential to enhance cytoskeletal organization and cell wall composition, thereby enhancing biomass production and growth in microgravity. Finally, understanding and manipulating the dysregulation of plastid gene transcription has further potential to address the goal of enhancing plant growth in the stressful conditions of microgravity.

Growth in spaceflight hardware results in alterations to the transcriptome and proteome.

The Biological Research in Canisters (BRIC) hardware has been used to house many biology experiments on both the Space Transport System (STS, commonly known as the space shuttle) and the International Space Station (ISS). However, microscopic examination of Arabidopsis seedlings by Johnson et al. (2015) indicated the hardware itself may affect cell morphology. The experiment herein was designed to assess the effects of the BRIC-Petri Dish Fixation Units (BRIC-PDFU) hardware on the transcriptome and proteome of Arabidopsis seedlings. To our knowledge, this is the first transcriptomic and proteomic comparison of Arabidopsis seedlings grown with and without hardware. Arabidopsis thaliana wild-type Columbia (Col-0) seeds were sterilized and bulk plated on forty-four 60 mm Petri plates, of which 22 were integrated into the BRIC-PDFU hardware and 22 were maintained in closed containers at Ohio University. Seedlings were grown for approximately 3 days, fixed with RNAlater® and stored at -80 °C prior to RNA and protein extraction, with proteins separated into membrane and soluble fractions prior to analysis. The RNAseq analysis identified 1651 differentially expressed genes; MS/MS analysis identified 598 soluble and 589 membrane proteins differentially abundant both at p < .05. Fold enrichment analysis of gene ontology terms related to differentially expressed transcripts and proteins highlighted a variety of stress responses. Some of these genes and proteins have been previously identified in spaceflight experiments, indicating that these genes and proteins may be perturbed by both conditions.

Transcriptome and proteome responses in RNAlater preserved tissue of Arabidopsis thaliana.

Tissue preservation is a minimal requirement for the success of plant RNA and protein expression studies. The standard of snap-freezing in liquid nitrogen is not always practical or possible. RNAlater, a concentrated solution of ammonium and cesium sulfates, has become a standard preservative in the absence of liquid nitrogen. Here, we demonstrate the effectiveness of RNAlater in preserving both RNA and proteins in Arabidopsis thaliana tissues for use in RNAseq and LC-MS/MS analysis of proteins. While successful in preserving plant material, a transcriptomic and proteomic response is evident. Specifically, 5770 gene transcripts, 84 soluble proteins, and 120 membrane-bound proteins were found to be differentially expressed at a log-fold change of ±1 (P ≤ 0.05). This response is mirrored in the abundance of post-translational modifications, with 23 of the 108 (21.3%) phosphorylated proteins showing altered abundance at a log-fold change of ±1 (P ≤ 0.05). While RNAlater is effective in preserving biological information, our findings warrant caution in its use for transcriptomic and proteomic experiments.

Proteomic approaches and their application to plant gravitropism.

Proteomics is a powerful technique that allows researchers a window into how an organism responds to a mutation, a specific environment, or at a distinct point during development by quantifying relative protein abundance and posttranslational modifications. Here, we describe methods for the proteomic analysis of Arabidopsis thaliana tissue. Extraction protocols are provided for isolation of soluble, plasma membrane, and tonoplast proteins. In addition, basic analysis and quality metrics for MS/MS data are discussed. The protocols outlined have the potential to unlock new avenues of research that are not possible through basic genetics or transcriptomic approaches. By combining proteomic information with known gene regulatory patterns, researchers can gain a complete picture of how molecular pathways, such as those required for gravitropism, are initiated, regulated, and terminated.

RNA Sequencing Analysis of the msl2msl3, crl, and ggps1 Mutants Indicates that Diverse Sources of Plastid Dysfunction Do Not Alter Leaf Morphology Through a Common Signaling Pathway.

Determining whether individual genes function in the same or in different pathways is an important aspect of genetic analysis. As an alternative to the construction of higher-order mutants, we used contemporary expression profiling methods to perform pathway analysis on several Arabidopsis thaliana mutants, including the mscS-like (msl)2msl3 double mutant. MSL2 and MSL3 are implicated in plastid ion homeostasis, and msl2msl3 double mutants exhibit leaves with a lobed periphery, a rumpled surface, and disturbed mesophyll cell organization. Similar developmental phenotypes are also observed in other mutants with defects in a range of other chloroplast or mitochondrial functions, including biogenesis, gene expression, and metabolism. We wished to test the hypothesis that the common leaf morphology phenotypes of these mutants are the result of a characteristic nuclear expression pattern that is generated in response to organelle dysfunction. RNA-Sequencing was performed on aerial tissue of msl2msl3 geranylgeranyl diphosphate synthase 1 (ggps1), and crumpled leaf (crl) mutants. While large groups of co-expressed genes were identified in pairwise comparisons between genotypes, we were only able to identify a small set of genes that showed similar expression profiles in all three genotypes. Subsequent comparison to the previously published gene expression profiles of two other mutants, yellow variegated 2 (var2) and scabra3 (sca3), failed to reveal a common pattern of gene expression associated with superficially similar leaf morphology defects. Nor did we observe overlap between genes differentially expressed in msl2msl3, crl, and ggps1 and a previously identified retrograde core response module. These data suggest that a common retrograde signaling pathway initiated by organelle dysfunction either does not exist in these mutants or cannot be identified through transcriptomic methods. Instead, the leaf developmental defects observed in these mutants may be achieved through a number of independent pathways.

Mutations in GERANYLGERANYL DIPHOSPHATE SYNTHASE 1 affect chloroplast development in Arabidopsis thaliana (Brassicaceae).

PREMISE OF THE STUDY: Within plastids, geranylgeranyl diphosphate synthase is a key enzyme in the isoprenoid biosynthetic pathway that catalyzes the formation of geranylgeranyl diphosphate, a precursor molecule to several biochemical pathways including those that lead into the biosynthesis of carotenoids and abscisic acid, prenyllipids such as the chlorophylls, and diterpenes such as gibberellic acid. • METHODS: We have identified mutants in the GERANYLGERANYL DIPHOSPHATE SYNTHASE 1 (GGPS1) gene, which encodes the major plastid-localized enzyme geranylgeranyl diphosphate synthase in Arabidopsis thaliana. • KEY RESULTS: Two T-DNA insertion mutant alleles (ggps1-2 and ggps1-3) were found to result in seedling-lethal albino and embryo-lethal phenotypes, respectively, indicating that GGPS1 is an essential gene. We also identified a temperature-sensitive leaf variegation mutant (ggps1-1) in A. thaliana that is caused by a point mutation. Total chlorophyll and carotenoid levels were reduced in ggps1-1 white tissues as compared with green tissues. Phenotypes typically associated with a reduction in gibberellic acid were not seen, suggesting that gibberellic acid biosynthesis is not noticeably altered in the mutant. In contrast to other variegated mutants, the ggps1-1 green sector photosynthetic rate was not elevated relative to wild-type tissues. Chloroplast development in green sectors of variegated leaves appeared normal, whereas cells in white sectors contained abnormal plastids with numerous electron translucent bodies and poorly developed internal membranes. • CONCLUSIONS: Our results indicate that GGPS1 is a key gene in the chlorophyll biosynthetic pathway.

Integration of Phot1, Phot2, and PhyB signalling in light-induced chloroplast movements.

In Arabidopsis thaliana, chloroplasts move towards the periclinal cell walls upon exposure to low blue light intensities and to anticlinal walls under high light. The regulation of these chloroplast movements involves members of both the phototropin and phytochrome families of photoreceptors. Examination of fluence-rate response dependencies in phot1 and phot2 mutants revealed that although both photoreceptors are capable of inducing chloroplast accumulation under low-light conditions, the signals from these photoreceptors appear to be antagonistic. Chloroplast movements in wild-type plants were intermediate between those of the single phot mutants, consistent with each operating through separate signalling cascades. Mutants in phot2 showed transient chloroplast avoidance responses upon exposure to intense blue light, and slow but sustained chloroplast avoidance under intense white light, indicating that in the absence of phot2, phot1 is capable of generating both a low and a high-light response signal. Mutations in phytochrome B (phyB) caused an enhanced avoidance response at intermediate and high light intensities. Examination of phyB, phot1phyB, and phot2phyB mutants indicated that this enhancement is caused by PhyB inhibition of the high-light avoidance response in wild-type plants. In addition, our results suggest that the inhibition by PhyB is not exclusive to either of the phot1 or phot2 signalling pathways.

Plastid movement impaired 2, a new gene involved in normal blue-light-induced chloroplast movements in Arabidopsis.

Chloroplasts move in a light-dependent manner that can modulate the photosynthetic potential of plant cells. Identification of genes required for light-induced chloroplast movement is beginning to define the molecular machinery that controls these movements. In this work, we describe plastid movement impaired 2 (pmi2), a mutant in Arabidopsis (Arabidopsis thaliana) that displays attenuated chloroplast movements under intermediate and high light intensities while maintaining a normal movement response under low light intensities. In wild-type plants, fluence rates below 20 micromol m(-2) s(-1) of blue light lead to chloroplast accumulation on the periclinal cell walls, whereas light intensities over 20 micromol m(-2) s(-1) caused chloroplasts to move toward the anticlinal cell walls (avoidance response). However, at light intensities below 75 micromol m(-2) s(-1), chloroplasts in pmi2 leaves move to the periclinal walls; 100 micromol m(-2) s(-1) of blue light is required for chloroplasts in pmi2 to move to the anticlinal cell walls, indicating a shift in the light threshold for the avoidance response in the mutant. The pmi2 mutation has been mapped to a gene that encodes a protein of unknown function with a large coiled-coil domain in the N terminus and a putative P loop. PMI2 shares sequence and structural similarity with PMI15, another unknown protein in Arabidopsis that, when mutated, causes a defect in chloroplast avoidance under high-light intensities.

Phytochrome modulation of blue light-induced chloroplast movements in Arabidopsis.

Photometric analysis of chloroplast movements in various phytochrome (phy) mutants of Arabidopsis showed that phyA, B, and D are not required for chloroplast movements because blue light (BL)-dependent chloroplast migration still occurs in these mutants. However, mutants lacking phyA or phyB showed an enhanced response at fluence rates of BL above 10 micromol m-2 s-1. Overexpression of phyA or phyB resulted in an enhancement of the low-light response. Analysis of chloroplast movements within the range of BL intensities in which the transition between the low- and high-light responses occur (1.5-15 micromol m-2 s-1) revealed a transient increase in light transmittance through leaves, indicative of the high-light response, followed by a decrease in transmittance to a value below that measured before the BL treatment, indicative of the low-light response. A biphasic response was not observed for phyABD leaves exposed to the same fluence rate of BL, suggesting that phys play a role in modulating the transition between the low- and high-light chloroplast movement responses of Arabidopsis.