Differences in osmotic adjustment, foliar abscisic acid dynamics, and stomatal regulation between an isohydric and anisohydric woody angiosperm during drought.

Species are often classified along a continuum from isohydric to anisohydric, with isohydric species exhibiting tighter regulation of leaf water potential through stomatal closure in response to drought. We investigated plasticity in stomatal regulation in an isohydric (Eucalyptus camaldulensis) and an anisohydric (Acacia aptaneura) angiosperm species subject to repeated drying cycles. We also assessed foliar abscisic acid (ABA) content dynamics, aboveground/belowground biomass allocation and nonstructural carbohydrates. The anisohydric species exhibited large plasticity in the turgor loss point (ΨTLP ), with plants subject to repeated drying exhibiting lower ΨTLP and correspondingly larger stomatal conductance at low water potential, compared to plants not previously exposed to drought. The anisohydric species exhibited a switch from ABA to water potential-driven stomatal closure during drought, a response previously only reported for anisohydric gymnosperms. The isohydric species showed little osmotic adjustment, with no evidence of switching to water potential-driven stomatal closure, but did exhibit increased root:shoot ratios. There were no differences in carbohydrate depletion between species. We conclude that a large range in ΨTLP and biphasic ABA dynamics are indicative of anisohydric species, and these traits are associated with exposure to low minimum foliar water potential, dense sapwood and large resistance to xylem embolism.

Contrasting regulation of leaf gas exchange of semi-arid tree species under repeated drought.

Predicting how plants respond to drought requires an understanding of how physiological mechanisms and drought response strategies occur, as these strategies underlie rates of gas exchange and productivity. We assessed the response of 11 plant traits to repeated experimental droughts in four co-occurring species of central Australia. The main goals of this study were to: (i) compare the response to drought between species; (ii) evaluate whether plants acclimated to repeated drought; and (iii) examine the degree of recovery in leaf gas exchange after cessation of drought. Our four species of study were two tree species and two shrub species, which field studies have shown to occupy different ecohydrological niches. The two tree species (Eucalyptus camaldulensis Dehnh. and Corymbia opaca (D.J.Carr & S.G.M.Carr) K.D.Hill & L.A.S.Johnson) had large reductions in stomatal conductance (gs) values, declining by 90% in the second drought. By contrast, the shrub species (Acacia aptaneura Maslin & J.E.Reid and Hakea macrocarpa A.Cunn. ex R.Br.) had smaller reductions gs in the second drought of 52 and 65%, respectively. Only A. aptaneura showed a physiological acclimatation to drought due to small declines in gs versus á´ªpd (0.08 slope) during repeated droughts, meaning they maintained higher rates of gs compared with plants that only experienced one final drought (0.19 slope). All species in all treatments rapidly recovered leaf gas exchange and leaf mass per area following drought, displaying physiological plasticity to drought exposure. This research refines our understanding of plant physiological responses to recurrent water stress, which has implications for modelling of vegetation, carbon assimilation and water use in semi-arid environments under drought.

A universal molecular control for DNA, mRNA and protein expression.

The expression of genes encompasses their transcription into mRNA followed by translation into protein. In recent years, next-generation sequencing and mass spectrometry methods have profiled DNA, RNA and protein abundance in cells. However, there are currently no reference standards that are compatible across these genomic, transcriptomic and proteomic methods, and provide an integrated measure of gene expression. Here, we use synthetic biology principles to engineer a multi-omics control, termed pREF, that can act as a universal molecular standard for next-generation sequencing and mass spectrometry methods. The pREF sequence encodes 21 synthetic genes that can be in vitro transcribed into spike-in mRNA controls, and in vitro translated to generate matched protein controls. The synthetic genes provide qualitative controls that can measure sensitivity and quantitative accuracy of DNA, RNA and peptide detection. We demonstrate the use of pREF in metagenome DNA sequencing and RNA sequencing experiments and evaluate the quantification of proteins using mass spectrometry. Unlike previous spike-in controls, pREF can be independently propagated and the synthetic mRNA and protein controls can be sustainably prepared by recipient laboratories using common molecular biology techniques. Together, this provides a universal synthetic standard able to integrate genomic, transcriptomic and proteomic methods.

Library adaptors with integrated reference controls improve the accuracy and reliability of nanopore sequencing.

Library adaptors are short oligonucleotides that are attached to RNA and DNA samples in preparation for next-generation sequencing (NGS). Adaptors can also include additional functional elements, such as sample indexes and unique molecular identifiers, to improve library analysis. Here, we describe Control Library Adaptors, termed CAPTORs, that measure the accuracy and reliability of NGS. CAPTORs can be integrated within the library preparation of RNA and DNA samples, and their encoded information is retrieved during sequencing. We show how CAPTORs can measure the accuracy of nanopore sequencing, evaluate the quantitative performance of metagenomic and RNA sequencing, and improve normalisation between samples. CAPTORs can also be customised for clinical diagnoses, correcting systematic sequencing errors and improving the diagnosis of pathogenic BRCA1/2 variants in breast cancer. CAPTORs are a simple and effective method to increase the accuracy and reliability of NGS, enabling comparisons between samples, reagents and laboratories, and supporting the use of nanopore sequencing for clinical diagnosis.

Night and day: Shrinking and swelling of stems of diverse mangrove species growing along environmental gradients.

Tree stems swell and shrink daily, which is thought to reflect changes in the volume of water within stem tissues. We observed these daily patterns using automatic dendrometer bands in a diverse group of mangrove species over five mangrove forests across Australia and New Caledonia. We found that mangrove stems swelled during the day and shrank at night. Maximum swelling was highly correlated with daily maxima in air temperature. Variation in soil salinity and levels of tidal inundation did not influence the timing of stem swelling over all species. Medium-term increases in stem circumference were highly sensitive to rainfall. We defoliated trees to assess the role of foliar transpiration in stem swelling and shrinking. Defoliated trees showed maintenance of the pattern of daytime swelling, indicating that processes other than canopy transpiration influence the temporary stem diameter increments, which could include thermal swelling of stems. More research is required to understand the processes contributing to stem shrinking and swelling. Automatic Dendrometer Bands could provide a useful tool for monitoring the response of mangroves to extreme climatic events as they provide high-frequency, long-term, and large-scale information on tree water status.

Synthetic microbe communities provide internal reference standards for metagenome sequencing and analysis.

The complexity of microbial communities, combined with technical biases in next-generation sequencing, pose a challenge to metagenomic analysis. Here, we develop a set of internal DNA standards, termed "sequins" (sequencing spike-ins), that together constitute a synthetic community of artificial microbial genomes. Sequins are added to environmental DNA samples prior to library preparation, and undergo concurrent sequencing with the accompanying sample. We validate the performance of sequins by comparison to mock microbial communities, and demonstrate their use in the analysis of real metagenome samples. We show how sequins can be used to measure fold change differences in the size and structure of accompanying microbial communities, and perform quantitative normalization between samples. We further illustrate how sequins can be used to benchmark and optimize new methods, including nanopore long-read sequencing technology. We provide metagenome sequins, along with associated data sets, protocols, and an accompanying software toolkit, as reference standards to aid in metagenomic studies.

Divergence in plant water-use strategies in semiarid woody species.

Partitioning of water resources amongst plant species within a single climate envelope is possible if the species differ in key hydraulic traits. We examined 11 bivariate trait relationships across nine woody species found in the Ti-Tree basin of central Australia. We found that species with limited access to soil moisture, evidenced by low pre-dawn leaf water potential, displayed anisohydric behaviour (e.g. large seasonal fluctuations in minimum leaf water potential), had greater sapwood density and lower osmotic potential at full turgor. Osmotic potential at full turgor was positively correlated with the leaf water potential at turgor loss, which was, in turn, positively correlated with the water potential at incipient stomatal closure. We also observed divergent behaviour in two species of Mulga, a complex of closely related Acacia species which range from tall shrubs to low trees and dominate large areas of arid and semiarid Australia. These Mulga species had much lower minimum leaf water potentials and lower specific leaf area compared with the other seven species. Finally, one species, Hakea macrocarpa A.Cunn ex.R.Br., had traits that may allow it to tolerate seasonal dryness (through possession of small specific leaf area and cavitation resistant xylem) despite exhibiting cellular water relations that were similar to groundwater-dependent species. We conclude that traits related to water transport and leaf water status differ across species that experience differences in soil water availability and that this enables a diversity of species to exist in this low rainfall environment.

Soil moisture controls on phenology and productivity in a semi-arid critical zone.

The Earth's Critical Zone, where physical, chemical and biological systems interact, extends from the top of the canopy to the underlying bedrock. In this study, we investigated soil moisture controls on phenology and productivity of an Acacia woodland in semi-arid central Australia. Situated on an extensive sand plain with negligible runoff and drainage, the carry-over of soil moisture content (θ) in the rhizosphere enabled the delay of phenology and productivity across seasons, until conditions were favourable for transpiration of that water to prevent overheating in the canopy. Storage of soil moisture near the surface (in the top few metres) was promoted by a siliceous hardpan. Pulsed recharge of θ above the hardpan was rapid and depended upon precipitation amount: 150mm storm(-1) resulted in saturation of θ above the hardpan (i.e., formation of a temporary, discontinuous perched aquifer above the hardpan in unconsolidated soil) and immediate carbon uptake by the vegetation. During dry and inter-storm periods, we inferred the presence of hydraulic lift from soil storage above the hardpan to the surface due to (i) regular daily drawdown of θ in the reservoir that accumulates above the hardpan in the absence of drainage and evapotranspiration; (ii) the dimorphic root distribution wherein most roots were found in dry soil near the surface, but with significant root just above the hardpan; and (iii) synchronisation of phenology amongst trees and grasses in the dry season. We propose that hydraulic redistribution provides a small amount of moisture that maintains functioning of the shallow roots during long periods when the surface soil layer was dry, thereby enabling Mulga to maintain physiological activity without diminishing phenological and physiological responses to precipitation when conditions were favourable to promote canopy cooling.

Radiocarbon dating and wood density chronologies of mangrove trees in arid Western Australia.

Mangrove trees tend to be larger and mangrove communities more diverse in tropical latitudes, particularly where there is high rainfall. Variation in the structure, growth and productivity of mangrove forests over climatic gradients suggests they are sensitive to variations in climate, but evidence of changes in the structure and growth of mangrove trees in response to climatic variation is scarce. Bomb-pulse radiocarbon dating provides accurate dates of recent wood formation and tree age of tropical and subtropical tree species. Here, we used radiocarbon techniques combined with X-ray densitometry to develop a wood density chronology for the mangrove Avicennia marina in the Exmouth Gulf, Western Australia (WA). We tested whether wood density chronologies of A. marina were sensitive to variation in the Pacific Decadal Oscillation Index, which reflects temperature fluctuations in the Pacific Ocean and is linked to the instrumental rainfall record in north WA. We also determined growth rates in mangrove trees from the Exmouth Gulf, WA. We found that seaward fringing A. marina trees (~10 cm diameter) were 48 ± 1 to 89 ± 23 years old (mean ± 1 σ) and that their growth rates ranged from 4.08 ± 2.36 to 5.30 ± 3.33 mm/yr (mean ± 1 σ). The wood density of our studied mangrove trees decreased with increases in the Pacific Decadal Oscillation Index. Future predicted drying of the region will likely lead to further reductions in wood density and their associated growth rates in mangrove forests in the region.

The anatomical basis of the link between density and mechanical strength in mangrove branches.

Tree branches are important as they support the canopy, which controls photosynthetic carbon gain and determines ecological interactions such as competition with neighbours. Mangrove trees are subject to high wind speeds, strong tidal flows and waves that can damage their branches. The survival and establishment of mangroves partly depend on the structural and mechanical characteristics of their branches. In addition, mangroves are exposed to soils that vary in salinity. Highly saline conditions can increase the tension in the water column, imposing mechanical stresses on the xylem vessels. Here, we investigated how mechanical strength, assessed as the modulus of elasticity (MOE) and the modulus of rupture (MOR), and density relate to the anatomical characteristics of intact mangrove branches from southeast Queensland and whether the mechanical strength of branches varies among mangrove species. Mechanical strength was positively correlated with density of mangrove intact branches. Mechanical strength (MOE) varied among species, with Avicennia marina (Forssk.) Vierh. branches having the highest mechanical strength (2079±176MPa), and Rhizophora stylosa Griff. and Bruguiera gymnorrhiza (L.) Savigny ex Lam. and Poiret having the lowest mechanical strength (536.8±39.2MPa in R. stylosa and 554±58.2MPa in B. gymnorrhiza). High levels of mechanical strength were associated with reductions in xylem vessel lumen area, pith content and bark content, and positively associated with increases in fibre wall thickness. The associations between mechanical strength and anatomical characteristics in mangrove branches suggest trade-offs between mechanical strength and water supply, which are linked to tree growth and survival.