Jazmine L. Humphreys

Time series omics experiments are critical for studying a range of biological processes, such as cell differentiation and developmental programs or responses to pathogens and environmental cues. While statistical tools for differential analysis across static conditions have matured, a comparable comprehensive methodology is lacking for time series data. Here, we introduce cpam, a novel method and user-friendly R package that performs temporal differential analysis of omics time series data, including pseudobulked single-cell sequence data. Powerful features include change-point detection and shape-constrained temporal trend estimation to allocate omics data into similar clusters. The software handles case-only and case-control designs, incorporates quantification uncertainty, and provides an interactive interface with customisable visualisations, offering graphical and statistical insight into molecular processes. Performance evaluation shows that cpam outperforms existing time series methods in terms of control of the false discovery rate versus power to detect temporal changes and accurate changepoint estimation. Application to published data illustrates RNA isoform-level modelling with high-resolution clustering during human embryogenesis and the identification of 910 novel genes that respond to excess-light in Arabidopsis.

Themeda triandra (family Andropogoneae) is a pan-continental grass occurring throughout Australia. Recognising its remarkable adaptive capacity, we assembled the nuclear genome sequence of a diploid accession from eastern Australia, finding all ten chromosomes of the 755 Mb assembly highly syntenic with those of Sorghum bicolor . Genotyping and cytometry of range-wide accessions suggested several historical radiation events, with polyploids dominating in arid regions, apart from a notable diploid lineage from the arid north-west of Australia (PAN). A detailed comparison of PAN with two diploids from temperate south-eastern Australia through whole-genome resequencing revealed extensive copy number variation and polymorphism, with changes in genes for heat-shock proteins and flowering regulation reflecting their environmental origin. Exploration of the rich genetic diversity in T. triandra with respect to environmental adaptation is expected to benefit grassland management programs and enable introgression of novel genes into Andropogoneae crops for climate resilience.

Plant genes can each produce multiple RNA isoforms, but little is known about when and how they are produced, or their functions. We conducted a detailed time course analysis of rapid changes in abundance of RNA isoforms in response to transient water deficit in Arabidopsis thaliana seedlings. We identified 95,104 transcripts in total, 21,935 of which were differentially expressed. Strikingly, 1,258 differentially expressed genes were identified only by means of RNA isoform analysis and would have been missed by conventional analysis. Several hundred genes exhibited a very rapid and reversible switch in the most abundant RNA isoform, with timings corresponding to defined changes in seedling water potential. In most cases it is predicted that RNA isoform switching would generate new protein products. Many of these genes encoded proteins of RNA metabolism and splicing, while others potentially function more directly in the response to water deficit. We propose a model in which very rapid RNA processing mechanisms come into play within minutes of imposition of the stress, changing the RNA isoform population including RNAs that encode components of the RNA processing apparatus itself. These changes together with subsequent changes in transcription facilitate further changes in the RNA isoform population as part of the stress and recovery responses. More broadly we propose that responses to abiotic stress involve substantial changes in gene function through the production of RNA isoforms, and we present a detailed approach to identifying such genome-wide RNA isoform switching.

Liverworts can clonally propagate by producing compact shoot structures called gemmae, which develop within basket-like structures known as gemma cups. It was previously reported in Marchantia nepalensis that carbon availability promotes gemma cup formation. However, the mechanisms by which carbon availability controls this process remains largely unexplored. To address this knowledge gap, we investigated how carbon promotes gemma cup formation using Marchantia polymorpha as a model species. Through a series of pharmacological and genetic experiments, we found that carbon availability promotes gemma cup formation by inducing the cytokinin pathway, thereby increasing the expression of MpGCAM1 and MpSTG, which encode two transcription factors involved in forming the basal floor of gemma cups. Indeed, our data show that cytokinins accumulate in marchantia thallus in response to sucrose and to high light treatments. In addition, constitutive induction of cytokinin signalling could overcome the repressive effect of low sucrose on gemma cup formation, whereas suppression of this hormonal pathway led to inhibition of sucrose-induced gemma cup formation. Furthermore, our results indicate that sucrose can induce gemma cup formation independently of KAI2A and MAX2, two molecular components of karrikin signalling known to control this developmental process by inducing cytokinin synthesis. Interestingly, in flowering plants, carbon availability also promotes cytokinin accumulation to induce axillary bud outgrowth, a process involving the transcription factors AtRAX and AtLOF1, the Arabidopsis thaliana orthologues of MpGCAM1 and MpSTG, respectively. Collectively, these observations indicate that the interactions between carbon and cytokinins are critical for the developmental plasticity of land plants in response to their environment.

Summary: Plants prioritise water allocation to tissues that support their survival and reproduction during drought, but little is known about their tissue‐specific drought tolerance mechanisms. In the case of basal buds, the extent to which they are hydraulically connected to the stem is unclear, as are their drought resistance mechanisms. To address these questions in the herbaceous annual Pisum sativum L. (cv. Torsdag), we used optical dendrometry to monitor the expansion and contraction of basal buds and their adjacent stems during their diurnal cycle and longer‐term during drought. We also measured water potential and osmotic potential of buds and stems. Dimensions of buds and stems oscillated synchronously throughout their diurnal cycle, indicating a functional hydraulic connection. Buds maintained turgor during drought by osmotic adjustment (OA) to maintain a pressure differential with the main plant xylem. Thus, immediate outgrowth of buds occurred upon rewatering. We conclude that OA protects buds during drought, allowing them to retain water and viability. This strategy enables growth and flowering of lateral branches postdrought, even if the primary shoot is damaged. We expect this mechanism to play a significant role in a wide range of annuals during drought, including many crop species.

Time series omics experiments are critical for the study of a wide range of biological processes such as cell differentiation and developmental programs or responses to pathogens and environmental cues. While statistical tools for differential analysis across static conditions have matured, comparable comprehensive methodology is lacking for time series data. Here, we introduce cpam , a novel time series method and user-friendly software that performs temporal differential analysis of omics data for case-only or case-control time series, incorporating quantification uncertainty. Powerful features of cpam include changepoint detection and shape-constrained temporal trend estimation to allocate omics data into similar clusters. Performance evaluation shows that cpam outperforms existing time series methods in terms of control of the false discovery rate versus power to detect temporal changes and accurate changepoint estimation. In particular, we find that trend-based models provide superior performance compared to pairwise comparisons, even with as few as six time points. The software provides an interactive interface with modern customizable visualisations, offering both graphical and statistical insight into complex molecular processes. Application to published transcriptome data illustrates RNA isoform-level modelling together with high resolution clustering during human embryogenesis and the identification of 910 novel genes differentially expressed in response to excess light in Arabidopsis. This method has the potential for application to a range of omics data, including single-cell analyses via pseudobulked data, providing the analytical power to reveal complex temporal patterns of gene expression in any biological system.

Summary: The phytohormone strigolactone (SL) inhibits shoot branching, whereas the signalling metabolite trehalose 6‐phosphate (Tre6P) promotes branching. How Tre6P and SL signalling may interact and which molecular mechanisms might be involved remains largely unknown. Transcript profiling of Arabidopsis SL mutants revealed a cluster of differentially expressed genes highly enriched in the Tre6P pathway compared with wild‐type (WT) plants or brc1 mutants. Tre6P‐related genes were also differentially expressed in axillary buds of garden pea (Pisum sativum) SL mutants. Tre6P levels were elevated in the SL signalling mutant more axillary (max) growth 2 compared with other SL mutants or WT plants indicating a role of MAX2‐dependent SL signalling in regulating Tre6P levels. A transgenic approach to increase Tre6P levels demonstrated that all SL mutant lines and brc1 flowered earlier, showing all of these mutants were responsive to Tre6P. Elevated Tre6P led to increased branching in WT plants but not in max2 and max4 mutants, indicating some dependency between the SL pathway and Tre6P regulation of shoot branching. By contrast, elevated Tre6P led to an enhanced branching phenotype in brc1 mutants indicating independence between BRC1 and Tre6P. A model is proposed whereby SL signalling represses branching via Tre6P and independently of the BRC1 pathway.

SUMMARY: Strigolactones (SL) function as plant hormones in control of multiple aspects of plant development, mostly via the regulation of gene expression. Immediate early‐gene regulation by SL remains unexplored due to difficulty in dissecting early from late gene expression responses to SL. We used synthetic SL, rac ‐GR24 treatment of protoplasts and RNA‐seq to explore early SL‐induced changes in gene expression over time (5–180 minutes) and discovered rapid, dynamic and SL receptor D14‐dependent regulation of gene expression in response to rac ‐GR24. Importantly, we discovered a significant dependence of SL signalling on chromatin remodelling processes, as the induction of a key SL‐induced transcription factor BRANCHED1 requires the SWI/SNF chromatin remodelling ATPase SPLAYED (SYD) and leads to upregulation of a homologue SWI/SNF ATPase BRAHMA . ATAC‐seq profiling of genome‐wide changes in chromatin accessibility in response to rac ‐GR24 identified large‐scale changes, with over 1400 differentially accessible regions. These changes in chromatin accessibility often precede transcriptional changes and are likely to harbour SL cis ‐regulatory elements. Importantly, we discovered that this early and extensive modification of the chromatin landscape also requires SYD. This study, therefore, provides evidence that SL signalling requires regulation of chromatin accessibility, and it identifies genomic locations harbouring likely SL cis ‐regulatory sequences.

The ontogenetic regulation of shoot branching allows plants to adjust their architecture in accordance with the environment. This process is due to the regulation of axillary bud outgrowth into branches, which can be induced by increasing sugar availability to the buds through decapitation of the shoot tip. Different sugar signalling components have been identified in the induction of shoot branching. However, the molecular components that maintain bud dormancy in response to sugar starvation remain largely unknown. Here, we show at the genetic level that basic leucine zipper 11 (bZIP11), a transcription factor that plays important roles in response to sugar starvation in plants, inhibits shoot branching in Arabidopsis thaliana . Physiology experiments demonstrated that bZIP11 protein levels are decreased by decapitation. Molecular and genetic evidence suggests that bZIP11 acts in a negative feedback loop with trehalose 6-phosphate (Tre6P), a sugar signal that promotes shoot branching. Our data also suggest that the central energy sensor SUCROSE NON-FERMENTING 1 RELATED KINASE1 (SnRK1), alleviates the inhibitory effect of Tre6P on bZIP11 protein accumulation and inhibits shoot branching. Altogether, these data provide a working model that involves bZIP11, Tre6P and SnRK1 in the regulation of shoot branching.