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Background and AimsThe leaf economic spectrum (LES) is related to dry mass and nutrient investments towards photosynthetic processes and leaf structures, and to the duration of returns on those investments (leaf lifespan, LL). Phosphorus (P) is a key limiting nutrient for plant growth, yet it is unclear how the allocation of leaf P among different functions is coordinated with the LES. We addressed this question among 12 evergreen woody species co-occurring on P-impoverished soils in south-eastern Australia.MethodsLeaf ‘economic’ traits, including LL, leaf mass per area (LMA), light-saturated net photosynthetic rate per mass (Amass), dark respiration rate, P concentration ([Ptotal]), nitrogen concentration, and P resorption, were measured for three pioneer and nine non-pioneer species. Leaf P was separated into five functional fractions: orthophosphate P (Pi), metabolite P (PM), nucleic acid P (PN), lipid P (PL), and residual P (PR; phosphorylated proteins and unidentified compounds that contain P).ResultsLL was negatively correlated with Amass and positively correlated with LMA, representing the LES. Pioneers occurred towards the short-LL end of the spectrum and exhibited higher [Ptotal] than non-pioneer species, primarily associated with higher concentrations of Pi, PN and PL. There were no significant correlations between leaf P fractions and LL or LMA, while Amass was positively correlated with the concentration of PR.ConclusionsAllocation of leaf P to different fractions varied substantially among species. This variation was partially associated with the LES, which may provide a mechanism underlying co-occurrence of species with different ecological strategies under P limitation.

Key messageA wild relative of rice from the Australian savannah was compared with cultivated rice, revealing thermotolerance in growth and photosynthetic processes and a more robust carbon economy in extreme heat.Above ~ 32 °C, impaired photosynthesis compromises the productivity of rice. We compared leaf tissues from heat-tolerant wild rice (Oryza australiensis) with temperate-adapted O. sativa after sustained exposure to heat, as well as diurnal heat shock. Leaf elongation and shoot biomass in O. australiensis were unimpaired at 45 °C, and soluble sugar concentrations trebled during 10 h of a 45 °C shock treatment. By contrast, 45 °C slowed growth strongly in O. sativa. Chloroplastic CO2 concentrations eliminated CO2 supply to chloroplasts as the basis of differential heat tolerance. This directed our attention to carboxylation and the abundance of the heat-sensitive chaperone Rubisco activase (Rca) in each species. Surprisingly, O. australiensis leaves at 45 °C had 50% less Rca per unit Rubisco, even though CO2 assimilation was faster than at 30 °C. By contrast, Rca per unit Rubisco doubled in O. sativa at 45 °C while CO2 assimilation was slower, reflecting its inferior Rca thermostability. Plants grown at 45 °C were simultaneously exposed to 700 ppm CO2 to enhance the CO2 supply to Rubisco. Growth at 45 °C responded to CO2 enrichment in O. australiensis but not O. sativa, reflecting more robust carboxylation capacity and thermal tolerance in the wild rice relative.

Next-generation sequencing demands high-quality nucleic acid, yet isolating DNA and RNA is often challenging, particularly from plant tissues. Despite advances in developing various kits and reagents, these products are tailored to isolation of nucleic acid from model plant tissues. Here we introduce a universal lysis buffer to separate nucleic acid from various plant species, including recalcitrant plants, to facilitate molecular analyses, such as quantitative PCR (qPCR), transcriptomics, and whole-genome sequencing (WGS). The protocol is a modification of the original CTAB methods, which leads to nucleic acid isolation from many plant species, including monocots and eudicots. The lysis buffer consists of hexadecyltrimethylammonium bromide (CTAB), sodium chloride (NaCl), Tris base, ethylenediaminetetraacetic acid (EDTA) and β-mercaptoethanol (βME). The modified CTAB method enables the isolation of nucleic acid from small amounts of plant tissues (e.g., 15–100 mg) in a timely manner, which is well-suited for a large number of samples and also when adequate sample collection is a limiting factor. The protocol isolates not only DNA from various plant species but also RNA. This makes it highly effective for molecular analyses compared to previously described CTAB methods optimised for DNA isolation. The appropriate concentration of the components enables high-quality DNA and RNA isolation from plant tissues simultaneously. Additionally, this protocol is compatible with commercially available columns. For DNA and RNA to be qualified for next-generation sequencing platforms, the protocol is supplemented with columns to purify either DNA or RNA from the same tissue to meet high standards for sequencing analyses. This protocol provides an ideal approach to overcome potential obstacles in isolating high-quality DNA or RNA from a wide range of plant species for downstream molecular analysis.

The mechanistic basis of tolerance to heat stress was investigated in Oryza sativa and two wild rice species, Oryza meridionalis and Oryza australiensis. The wild relatives are endemic to the hot, arid Australian savannah. Leaf elongation rates and gas exchange were measured during short periods of supraoptimal heat, revealing species differences. The Rubisco activase (RCA) gene from each species was sequenced. Using expressed recombinant RCA and leaf-extracted RCA, the kinetic properties of the two isoforms were studied under high temperatures. Leaf elongation was undiminished at 45°C in O. australiensis. The net photosynthetic rate was almost 50% slower in O. sativa at 45°C than at 28°C, while in O. australiensis it was unaffected. Oryza meridionalis exhibited intermediate heat tolerance. Based on previous reports that RCA is heat-labile, the Rubisco activation state was measured. It correlated positively with leaf elongation rates across all three species and four periods of exposure to 45°C. Sequence analysis revealed numerous polymorphisms in the RCA amino acid sequence from O. australiensis. The O. australiensis RCA enzyme was thermally stable up to 42°C, contrasting with RCA from O. sativa, which was inhibited at 36°C. We attribute heat tolerance in the wild species to thermal stability of RCA, enabling Rubisco to remain active.

36 I. 36 II. 37 III. 44 IV. 49 V. 53 54 References 55 SUMMARY: Anoxia tolerance in plants is distinguished by direction of the sparse supply of energy to processes crucial to cell maintenance and sometimes to growth, as in rice seedlings. In anoxic rice coleoptiles energy is used to synthesise proteins, take up K⁺, synthesise cell walls and lipids, and in cell maintenance. Maintenance of electrochemical H⁺gradients across the tonoplast and plasma membrane is crucial for solute compartmentation and thus survival. These gradients sustain some H⁺‐solute cotransport and regulate cytoplasmic pH. Pyrophosphate (PPᵢ), the alternative energy donor to ATP, allows direction of energy to the vacuolar H⁺‐PPᵢase, sustaining H⁺gradients across the tonoplast. When energy production is critically low, operation of a biochemical pHstat allows H⁺‐solute cotransport across plasma membranes to continue for at least for 18 h. In active (e.g. growing) cells, PPᵢproduced during substantial polymer synthesis allows conversion of PPᵢto ATP by PPᵢ‐phosphofructokinase (PFK). In quiescent cells with little polymer synthesis and associated PPᵢformation, the PPᵢrequired by the vacuolar H⁺‐PPᵢase and UDPG pyrophosphorylase involved in sucrose mobilisation via sucrose synthase might be produced by conversion of ATP to PPᵢthrough reversible glycolytic enzymes, presumably pyruvate orthophosphate dikinase. These hypotheses need testing with species characterised by contrasting anoxia tolerance.

Oryza meridionalis Ng. is a wild relative of Oryza sativa L. found throughout northern Australia where temperatures regularly exceed 35 °C in the monsoon growing season. Heat tolerance in O. meridionalis was established by comparing leaf elongation and photosynthetic rates at 45 °C with plants maintained at 27 °C. By comparison with O. sativa ssp. japonica cv. Amaroo, O. meridionalis was heat tolerant. Elongation rates of the third leaf of O. meridionalis declined by 47% over 24 h at 45 °C compared with a 91% decrease for O. sativa. Net photosynthesis was significantly higher in O. sativa at 27 °C whereas the two species had the same assimilation rates at 45 °C. The leaf proteome and expression levels of individual heat-responsive genes provided insight into the heat response of O. meridionalis. After 24 h of heat exposure, many enzymes involved in the Calvin Cycle were more abundant, while mRNA of their genes generally decreased. Ferredoxin-NADP(H) oxidoreductase, a key enzyme in photosynthetic electron transport had both reduced abundance and gene expression, suggesting light reactions were highly susceptible to heat stress. Rubisco activase was strongly up-regulated after 24 h of heat, with the large isoform having the largest relative increase in protein abundance and a significant increase in gene expression. The protective proteins Cpn60, Hsp90, and Hsp70 all increased in both protein abundance and gene expression. A thiamine biosynthesis protein (THI1), previously shown to act protectively against stress, increased in abundance during heat, even as thiamine levels fell in O. meridionalis.

Genes encoding thermostable variants of the photosynthesis heat-labile protein Rubisco activase (Rca) from a wild relative were overexpressed in domesticated rice ( ). Proteomics was used to quantify the abundance of Rca (Rca- ) in the resulting plants. Plants were grown to maturity in growth rooms and from early tillering until immediately prior to anthesis, they were exposed to daytime maximum temperatures of 28, 40, and 45°C and constant night temperatures of 22°C. Non-destructive measurements of leaf elongation and photosynthesis were used to compare the segregant with a transfected line in which 19% of its total Rca content was the recombinant Rca (T- -19). Height, fresh mass, panicle number, seed set, and seed number were measured at final harvest. Traits at maturity after heat stress at 45°C correlated strongly with recombinant protein abundance. Seed number was far the most responsive trait to an increase in Rca- abundance, improving by up to 150%. Leaf elongation rates ( ) and tiller number were significantly greater in the transformed plants in the first two weeks of exposure to 45°C but tiller numbers later became equal in the two genotypes. Gas exchange measurements showed that T- -19 had faster light induction of photosynthesis but not significantly higher CO assimilation rates, indicating that the carbon gain that resulted in large yield improvement after growth at 45°C was not strongly correlated with an instantaneous measurement of steady-state photosynthesis. When plants were grown at 40°C daytime maximum, there was no improvement in the final biomass, panicle or seed number when compared with 28°C, indicating that the threshold for heat damage and beneficial effects of the thermostable Rca recombinant protein was between 40 and 45°C, which corresponded to leaf temperatures in the range 38-42°C. The results suggest that the thermotolerant form of Rca from was sufficient to enhance carbohydrate accumulation and storage by rice over the life of the plant, dramatically improving yields after exposure to heat throughout the vegetative phase.

Oryza australiensis is a wild rice native to monsoonal northern Australia. The International Oryza Map Alignment Project emphasises its significance as the sole representative of the EE genome clade. Assembly of the O. australiensis genome has previously been challenging due to its high Long Terminal Repeat (LTR) retrotransposon (RT) content. Oxford Nanopore long reads were combined with Illumina short reads to generate a high-quality ~ 858 Mbp genome assembly within 850 contigs with 46× long read coverage. Reference-guided scaffolding increased genome contiguity, placing 88.2% of contigs into 12 pseudomolecules. After alignment to the Oryza sativa cv. Nipponbare genome, we observed several structural variations. PacBio Iso-Seq data were generated for five distinct tissues to improve the functional annotation of 34,587 protein-coding genes and 42,329 transcripts. We also report SNV numbers for three additional O. australiensis genotypes based on Illumina re-sequencing. Although genetic similarity reflected geographical separation, the density of SNVs also correlated with our previous report on variations in salinity tolerance. This genome re-confirms the genetic remoteness of the O. australiensis lineage within the O. officinalis genome complex. Assembly of a high-quality genome for O. australiensis provides an important resource for the discovery of critical genes involved in development and stress tolerance.

Models of tree responses to climate typically project that elevated atmospheric CO₂ concentration (eCₐ) will reduce drought impacts on forests. We tested one of the mechanisms underlying this interaction, the ‘low Cᵢ effect’, in which stomatal closure in drought conditions reduces the intercellular CO₂ concentration (Cᵢ), resulting in a larger relative enhancement of photosynthesis with eCₐ, and, consequently, a larger relative biomass response. We grew two Eucalyptus species of contrasting drought tolerance at ambient and elevated Cₐ for 6–9 months in large pots maintained at 50% (drought) and 100% field capacity. Droughted plants did not have significantly lower Cᵢ than well‐watered plants, which we attributed to long‐term changes in leaf area. Hence, there should not have been an interaction between eCₐ and water availability on biomass, and we did not detect one. The xeric species did have higher Cᵢ than the mesic species, indicating lower water‐use efficiency, but both species exhibited similar responses of photosynthesis and biomass to eCₐ, owing to compensatory differences in the photosynthetic response to Cᵢ. Our results demonstrate that long‐term acclimation to drought, and coordination among species traits may be important for predicting plant responses to eCₐ under low water availability.

BackgroundSoil salinity is widespread in rice-producing areas globally, restricting both vegetative growth and grain yield. Attempts to improve the salt tolerance of Asian rice, Oryza sativa—the most salt sensitive of the major cereal crops—have met with limited success, due to the complexity of the trait and finite variation in salt responses among O. sativa lines. Naturally occurring variation among the more than 20 wild species of the Oryza genus has great potential to provide breeders with novel genes to improve resistance to salt. Here, through two distinct screening experiments, we investigated variation in salinity tolerance among accessions of two wild rice species endemic to Australia, O. meridionalis and O. australiensis, with O. sativa cultivars Pokkali and IR29 providing salt-tolerant and sensitive controls, respectively.ResultsRice plants were grown on soil supplemented with field-relevant concentrations of NaCl (0, 40, 80, and 100 mM) for 30 d, a period sufficient to reveal differences in growth and physiological traits. Two complementary screening approaches were used: destructive phenotyping and high-throughput image-based phenotyping. All genotypes displayed clear responses to salt treatment. In the first experiment, both salt-tolerant Pokkali and an O. australiensis accession (Oa-VR) showed the least reduction in biomass accumulation, SES score and chlorophyll content in response to salinity. Average shoot Na+/K+ values of these plants were the lowest among the genotypes tested. In the second experiment, plant responses to different levels of salt stress were quantified over time based on projected shoot area calculated from visible red-green-blue (RGB) and fluorescence images. Pokkali grew significantly faster than the other genotypes. Pokkali and Oa-VR plants displayed the same absolute growth rate under 80 and 100 mM, while Oa-D grew significantly slower with the same treatments. Oa-VR showed substantially less inhibition of growth in response to salinity when compared with Oa-D. Senescence was seen in Oa-D after 30 d treatment with 40 mM NaCl, while the putatively salt-tolerant Oa-VR had only minor leaf damage, even at higher salt treatments, with less than a 40% increase in relative senescence at 100 mM NaCl compared to 120% for Oa-VR.ConclusionThe combination of our two screening experiments uncovered striking levels of salt tolerance diversity among the Australian wild rice accessions tested and enabled analysis of their growth responses to a range of salt levels. Our results validate image-based phenotyping as a valuable tool for quantitative measurement of plant responses to abiotic stresses. They also highlight the potential of exotic germplasm to provide new genetic variation for salinity tolerance in rice.