Regulation of desiccation tolerance in Xerophyta seedlings and leaves

A small, diverse group of angiosperms known as resurrection plants display vegetative desiccation tolerance and can survive loss of up to 95% of cellular water, a feat only seen in the seeds and pollen of other angiosperms. Xerophyta humilis is a resurrection plant native to Southern Africa that has been the target of previous transcriptomic and proteomic studies into the mechanisms of plant desiccation tolerance. The aim of this study was to investigate the hypothesis that vegetative desiccation tolerance is derived from the networks that control desiccation tolerance in seeds and germinating seedlings in angiosperms, particularly the epigenetically silenced seed maturation genes. Germinating seedlings of X. humilis and the related resurrection plant X. viscosa were found to be VDT from the earliest stages of germination, and exhibited the characteristic vegetative trait of poikilochlorophylly as seen in mature leaves. The X. humilis desiccation transcriptome comprising 76,768 distinct gene clusters was successfully assembled from sequencing samples at five relative water contents (100%, 80%, 60%, 40% and 5%) to identify the networks activated in response to water loss. Desiccation was associated with successive waves of transcription factor induction, as well as widespread down-regulation of histone modification enzymes. Many seed-specific genes, such as late embryogenesis abundant (LEA) proteins, seed storage proteins and oleosins, were induced in vegetative tissue. LEA transcripts in particular were highly up-regulated during desiccation, and the large number of distinct LEA transcripts (over 150) suggests possible LEA gene expansion in Xerophyta compared to desiccation-sensitive plants. Components of the PYL/SnRK2/ABF ABA-signalling pathway were also induced, although the ABF transcription factors activated in response to desiccation were most similar to those induced by drought in A. thaliana rather than seed maturation. Of the canonical seed master regulators (such as the LEC1/ABI3/FUS3/LEC2 network and ABI5) only three ABI3 transcripts were expressed, all of which encoded proteins lacking the seed motif-binding B3-domain. The results of this study suggest that vegetative desiccation tolerance in X. humilis is not associated with re-activation of seed master regulators in vegetative tissue, but may instead involve activation of seed genes by vegetative drought response regulators

The window of desiccation tolerance shown by early-stage germinating seedlings remains open in the resurrection plant, Xerophyta viscosa

Resurrection plants are renowned for their vegetative desiccation tolerance (DT). While DT in vegetative tissues is rare in angiosperms, it is ubiquitous in mature orthodox seeds. During germination, seedlings gradually lose DT until they pass a point of no return, after which they can no longer survive dehydration. Here we investigate whether seedlings of the resurrection plant Xerophyta viscosa ever lose the capacity to establish DT. Seedlings from different stages of germination were dehydrated for 48 hours and assessed for their ability to recover upon rehydration. While a transient decline in the ability of X. viscosa seedlings to survive dehydration was observed, at no point during germination was the ability to re-establish DT completely lost in all seedlings. Pre-treatment of seedlings with PEG or sucrose reduced this transient decline, and improved the survival rate at all stages of germination. Additionally, we observed that the trait of poikilochlorophylly (or loss of chlorophyll) observed in adult X. viscosa leaves can be induced throughout seedling development. These results suggest that the window of DT seen in germinating orthodox seeds remains open in X. viscosa seedlings and that vegetative DT in Xerophyta species may have evolved from the ability to retain this program through to adulthood

Comparative Analysis of ROS Network Genes in Extremophile Eukaryotes

The reactive oxygen species (ROS) gene network, consisting of both ROS-generating and detoxifying enzymes, adjusts ROS levels in response to various stimuli. We performed a cross-kingdom comparison of ROS gene networks to investigate how they have evolved across all Eukaryotes, including protists, fungi, plants and animals. We included the genomes of 16 extremotolerant Eukaryotes to gain insight into ROS gene evolution in organisms that experience extreme stress conditions. Our analysis focused on ROS genes found in all Eukaryotes (such as catalases, superoxide dismutases, glutathione reductases, peroxidases and glutathione peroxidase/peroxiredoxins) as well as those specific to certain groups, such as ascorbate peroxidases, dehydroascorbate/monodehydroascorbate reductases in plants and other photosynthetic organisms. ROS-producing NADPH oxidases (NOX) were found in most multicellular organisms, although several NOX-like genes were identified in unicellular or filamentous species. However, despite the extreme conditions experienced by extremophile species, we found no evidence for expansion of ROS-related gene families in these species compared to other Eukaryotes. Tardigrades and rotifers do show ROS gene expansions that could be related to their extreme lifestyles, although a high rate of lineage-specific horizontal gene transfer events, coupled with recent tetraploidy in rotifers, could explain this observation. This suggests that the basal Eukaryotic ROS scavenging systems are sufficient to maintain ROS homeostasis even under the most extreme conditions

Xerophyta humilis raw transcriptome

Dataset for the article Vegetative desiccation tolerance in the resurrection plant Xerophyta humilis has not evolved through reactivation of the seed canonical LAFL regulatory network.Raw transcripts from Trinity de novo assembly of combined seed and leaf Xerophyta humilis samples. Transcript IDs are the Trinity transcript ID followed by the unique gene cluster ID if one is present (example: TRINITY_DN164427_c0_g1_i1|Xh025390c1). Gene cluster IDs denote sets of transcripts that passed RapClust filtering and mapped uniquely to the same genomic locus on the draft Xerophyta humilis genome (and thus represent putative isoforms or fragments of the same gene)

Xerophyta humilis representative sequences

Dataset for the article Vegetative desiccation tolerance in the resurrection plant Xerophyta humilis has not evolved through reactivation of the seed canonical LAFL regulatory network.Representative transcripts: representative sequence for each gene cluster (the transcript with the longest CDS, or longest transcript if no CDS was found). Representative proteins: coding sequences of the best ORF for each representative transcript. LAFL-ABF: contigs and protein-coding sequences for the LAFL and ABI5/ABF gene sequences discussed in the manuscript.</div

Xerophyta humilis normalised expression

Dataset for the article Vegetative desiccation tolerance in the resurrection plant Xerophyta humilis has not evolved through reactivation of the seed canonical LAFL regulatory network.Normalised expression values for each gene cluster (i.e. at the gene level) for both seed and leaf datasets (calculated independently, but displayed together). Adjusted p-value is shown for each tissue type only if the change in expression was significant (FDR padj seed | padj leaf | early maturation seed 1 | early maturation seed 2 | early maturation seed 3 | mid maturation seed 1 | mid maturation seed 2 | mid maturation seed 3 | dry seed 1 | dry seed 2 | dry seed 3 | 100% RWC leaf 1 | 100% RWC leaf 2 | 100% RWC leaf 3 | 80% RWC leaf 1 | 80% RWC leaf 2 | 80% RWC leaf 3 | 60% RWC leaf 1 | 60% RWC leaf 2 | 60% RWC leaf 3 | 40% RWC leaf 1 | 40% RWC leaf 2 | 40% RWC leaf 3 | 5% RWC leaf 1 | 5% RWC leaf 2 | 5% RWC leaf 3</div

Recovery of <i>X. viscosa</i> seedlings from dehydration.

<p>Representative images of three untreated <i>X. viscosa</i> seedlings 1 mm, 2.2 mm and 3 mm in length before and after dehydration and rehydration. The 1 mm seedling dehydrated without chlorophyll loss, but recovered and resumed growth rapidly after rehydration. A 2.2 mm seedling likewise failed to degrade chlorophyll, but did not survive the rehydration process. A 3 mm seedling displayed partial chlorophyll loss from its cotyledon during dehydration. The seedling survived with its meristem, primary leaf and leading end of the cotyledon intact; the primary root failed to survive, but two additional secondary roots emerged from the meristem. Anthocyanins accumulated in the photosynthetic tissue during rehydration of this seedling.</p

Pre-treatment with PEG or sucrose improves desiccation tolerance in germinating seedlings of <i>A. thaliana</i> and <i>X. viscosa</i>.

<p>(A) Mean survival rate (±SEM) of <i>A. thaliana</i> seedlings after 48 h desiccation at various stages of germination. Although survival improves with PEG and sucrose treatment, <i>A. thaliana</i> seedlings are unable to survive desiccation past the “root hairs visible” seedling stage. (B) Survival of germinating <i>X. viscosa</i> seedlings with or without PEG or sucrose pre-treatment, grouped by cotyledon size (±SEM). Seedlings pre-treated with either sucrose or PEG did not show a decline in desiccation tolerance compared to untreated seedlings. (C) Chlorophyll loss in <i>X. viscosa</i> sucrose-treated seedlings. Although the mean survival rate of sucrose-treated seedlings was relatively consistent across most stages, the incidence of chlorophyll loss increased steadily with increasing seedling size. Seeds per bin can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093093#pone.0093093.s003" target="_blank">Table S1</a>.</p

Desiccation tolerance in germinating seedlings of <i>X. viscosa</i> and <i>A. thaliana</i>.

<p>(A) Survival of germinating <i>X. viscosa</i> seedlings after 48 h desiccation, grouped by cotyledon length. Data shows the mean survival rate ±SEM as determined by bootstrapping. Representative line drawings of germinating seedlings are shown above. (B) Incidence of chlorophyll degradation in the surviving seedlings from Figure 2A. Black bars: survived and did not lose chlorophyll. Grey bars: survived and lost chlorophyll. The numbers of seeds per bin are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093093#pone.0093093.s003" target="_blank">Table S1</a>.</p

Pre-treatment of <i>X. viscosa</i> seedlings with PEG results in chlorophyll degradation.

<p>Representative images of an untreated, PEG-treated and sucrose-treated seedling undergoing dehydration and rehydration. An untreated seedling (MS media) lost chlorophyll and accumulated anthocyanins in response to dehydration, and survived rehydration. A PEG treated seedling completely degraded its chlorophyll in response to PEG priming (prior to dehydration), and recovered well after rehydration. A sucrose-treated seedling failed to lose chlorophyll during dehydration; however, the meristem, primary leaf and a small portion of the cotyledon recovered after rehydration nonetheless.</p