Transcript stability is an important determinant of its abundance and, consequently, translational output. Transcript destabilisation can be rapid and is well suited for modulating the cellular response. However, it is unclear the extent to which RNA stability is altered under changing environmental conditions in plants. We previously hypothesised that recovery-induced transcript destabilisation facilitated a phenomenon of rapid recovery gene downregulation (RRGD) in Arabidopsis thaliana (Arabidopsis) following light stress, based on mathematical calculations to account for ongoing transcription. Here, we test this hypothesis and investigate processes regulating transcript abundance and fate by quantifying changes in transcription, stability and translation before, during and after light stress. We adapt syringe infiltration to apply a transcriptional inhibitor to soil-grown plants in combination with stress treatments. Compared with measurements in juvenile plants and cell culture, we find reduced stability across a range of transcripts encoding proteins involved in RNA binding and processing. We also observe light-induced destabilisation of transcripts, followed by their stabilisation during recovery. We propose that this destabilisation facilitates RRGD, possibly in combination with transcriptional shut-off that was confirmed for HSP101, ROF1 and GOLS1. We also show that translation remains highly dynamic over the course of light stress and recovery, with a bias towards transcript-specific increases in ribosome association, independent of changes in total transcript abundance, after 30 min of light stress. Taken together, we provide evidence for the combinatorial regulation of transcription and stability that occurs to coordinate translation during light stress and recovery in Arabidopsis.

Canonical retrograde signalling comprises information transmission from organelles to the nucleus and in particular controls gene expression for organellar proteins. The need to re-assess this paradigm was suggested by discrepancies between de novo protein synthesis and transcript abundance in response to excess light. Here we uncover major components of a translation-dependent retrograde signalling pathway that first impacts translation and then transcription. The response realization depends on the kinases Mitogen-activated protein kinase 6 (MPK6) and Sucrose non-fermenting 1-related kinase (SnRK1) subunit, AKIN10. Global ribosome foot-printing revealed differential ribosome association of 951 transcripts within 10 min after transfer from low to high light. Despite predominant translational repression, 15 % of transcripts were increased in translation and enriched for chloroplast-localized photosynthetic proteins. About one third of these transcripts, including Stress associated proteins (SAP) 2 and 3, share regulatory motifs in their 5′-UTR that act as binding sites for glyceraldehyde-3-phosphate dehydrogenase (GAPC) and light responsive RNA binding proteins (RBPs). SAP2 and 3 are both translationally regulated and interact with the calcium sensor Calmodulin-like 49 (CML49), which promotes relocation to the nucleus inducing a translation-dependent nuclear stress response. Thus, translation-dependent retrograde signalling bifurcates to directly regulate a translational circuit of chloroplast proteins and simultaneously initiate a nuclear circuit synchronizing retrograde and anterograde response pathways, serving as a rapid mechanism for functional acclimation of the chloroplast.

We describe our efforts at developing a one-step quantitative reverse-transcription (qRT)-PCR protocol to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA directly from saliva samples, without RNA purification. We find that both heat and the presence of saliva impairs the ability to detect synthetic SARS-CoV-2 RNA. Buffer composition (for saliva dilution) was also crucial to effective PCR detection. Using the SG2 primer pair, designed by Sigma-Aldrich, we were able to detect the equivalent of 1.7×10 6 viral copies per mL of saliva after heat inactivation; approximately equivalent to the median viral load in symptomatic patients. This would make our assay potentially useful for rapid detection of high-shedding infected individuals. We also provide a comparison of the PCR efficiency and specificity, which varied considerably, across 9 reported primer pairs for SARS-CoV-2 detection. Primer pairs SG2 and CCDC-N showed highest specificity and PCR efficiency. Finally, we provide an alternate primer pair to use as a positive control for human RNA detection in SARS-CoV-2 assays, as we found that the widely used US CDC primers (targeting human RPP30 ) do not span an exon-exon junction and therefore does not provide an adequate control for the reverse transcription reaction.

Consequences of transcription out of bounds: a retrograde signal can trigger RNA Polymerase II read-through, upregulating the expression of downstream genes. In plants, the molecular function(s) of the nucleus-localized 5′-3′ EXORIBONUCLEASES (XRNs) are unclear; however, their activity is reported to have a significant effect on gene expression and SAL1-mediated retrograde signaling. Using parallel analysis of RNA ends, we documented a dramatic increase in uncapped RNA substrates of the XRNs in both sal1 and xrn2xrn3 mutants. We found that a major consequence of reducing SAL1 or XRN activity was RNA Polymerase II 3′ read-through. This occurred at 72% of expressed genes, demonstrating a major genome-wide role for the XRN-torpedo model of transcription termination in Arabidopsis (Arabidopsis thaliana). Read-through is speculated to have a negative effect on transcript abundance; however, we did not observe this. Rather, we identified a strong association between read-through and increased transcript abundance of tandemly orientated downstream genes, strongly correlated with the proximity (less than 1,000 bp) and expression of the upstream gene. We observed read-through in the proximity of 903 genes up-regulated in the sal1-8 retrograde signaling mutant; thus, this phenomenon may account directly for up to 23% of genes up-regulated in sal1-8. Using APX2 and AT5G43770 as exemplars, we genetically uncoupled read-through loci from downstream genes to validate the principle of read-through-mediated mRNA regulation, providing one mechanism by which an ostensibly posttranscriptional exoribonuclease that targets uncapped RNAs could modulate gene expression.

In plants, the molecular function(s) of the nucleus-localized 5'-3' EXORIBONUCLEASES (XRNs) are unclear; however, their activity is reported to have a significant effect on gene expression and SAL1-mediated retrograde signaling. Using parallel analysis of RNA ends, we documented a dramatic increase in uncapped RNA substrates of the XRNs in both sal1 and xrn2xrn3 mutants. We found that a major consequence of reducing SAL1 or XRN activity was RNA Polymerase II 3' read-through. This occurred at 72% of expressed genes, demonstrating a major genome-wide role for the XRN-torpedo model of transcription termination in Arabidopsis (Arabidopsis thaliana). Read-through is speculated to have a negative effect on transcript abundance; however, we did not observe this. Rather, we identified a strong association between read-through and increased transcript abundance of tandemly orientated downstream genes, strongly correlated with the proximity (less than 1,000 bp) and expression of the upstream gene. We observed read-through in the proximity of 903 genes up-regulated in the sal1-8 retrograde signaling mutant; thus, this phenomenon may account directly for up to 23% of genes up-regulated in sal1-8 Using APX2 and AT5G43770 as exemplars, we genetically uncoupled read-through loci from downstream genes to validate the principle of read-through-mediated mRNA regulation, providing one mechanism by which an ostensibly posttranscriptional exoribonuclease that targets uncapped RNAs could modulate gene expression.

Science is about asking questions but not all science courses provide students with opportunities to practice this essential skill. We give students ownership of the processes of asking and answering questions to help them take greater responsibility for their own learning and to better understand the process of science with its inherent uncertainty. Peer learning activities throughout the course embed multidirectional feedback within and between students and instructors. Students are our partners in the design and evaluation of exam questions and we learn from them as they rise to the challenge of identifying important information and applying it. The lab program is supported by peer assisted learning in which peer mentors partner with instructors to generate activities addressing the use of evidence and experimental design. While not all students engage as partners, those who do value these experiences and demonstrate they can use scientific content creatively and critically.

Stress recovery may prove to be a promising approach to increase plant performance and, theoretically, mRNA instability may facilitate faster recovery. Transcriptome (RNA-seq, qPCR, sRNA-seq, and PARE) and methylome profiling during repeated excess-light stress and recovery was performed at intervals as short as 3 min. We demonstrate that 87% of the stress-upregulated mRNAs analyzed exhibit very rapid recovery. For instance, HSP101 abundance declined 2-fold every 5.1 min. We term this phenomenon rapid recovery gene downregulation (RRGD), whereby mRNA abundance rapidly decreases promoting transcriptome resetting. Decay constants (k) were modeled using two strategies, linear and nonlinear least squares regressions, with the latter accounting for both transcription and degradation. This revealed extremely short half-lives ranging from 2.7 to 60.0 min for 222 genes. Ribosome footprinting using degradome data demonstrated RRGD loci undergo cotranslational decay and identified changes in the ribosome stalling index during stress and recovery. However, small RNAs and 5'-3' RNA decay were not essential for recovery of the transcripts examined, nor were any of the six excess light-associated methylome changes. We observed recovery-specific gene expression networks upon return to favorable conditions and six transcriptional memory types. In summary, rapid transcriptome resetting is reported in the context of active recovery and cellular memory.