Andrew Merchant

Non-invasive approaches for phenotyping of enhanced performance traits in bean

Plant phenotyping is an emerging discipline in plant biology. Quantitative measurements of functional and structural traits help to better understand gene-environment interactions and support breeding for improved resource use efficiency of important crops such as bean (Phaseolus vulgaris L.). Here we provide an overview of state-of-the-art phenotyping approaches addressing three aspects of resource use efficiency in plants: belowground roots, aboveground shoots and transport/allocation processes. We demonstrate the capacity of high-precision methods to measure plant function or structural traits non-invasively, stating examples wherever possible. Ideally, high-precision methods are complemented by fast and high-throughput technologies. High-throughput phenotyping can be applied in the laboratory using automated data acquisition, as well as in the field, where imaging spectroscopy opens a new path to understand plant function non-invasively. For example, we demonstrate how magnetic resonance imaging (MRI) can resolve root structure and separate root systems under resource competition, how automated fluorescence imaging (PAM fluorometry) in combination with automated shape detection allows for high-throughput screening of photosynthetic traits and how imaging spectrometers can be used to quantify pigment concentration, sun-induced fluorescence and potentially photosynthetic quantum yield. We propose that these phenotyping techniques, combined with mechanistic knowledge on plant structure-function relationships, will open new research directions in whole-plant ecophysiology and may assist breeding for varieties with enhanced resource use efficiency varieties.

Phloem sap and leaf δ13C, carbohydrates and amino acid concentrations in Eucalyptus globulus change systematically according to flooding and water deficit treatment

Phloem is a central conduit for the distribution of photoassimilate, nutrients, and signals among plant organs. A revised technique was used to collect phloem sap from small woody plants in order to assess changes in composition induced by water deficit and flooding. Bled phloem sap δ13C and sugar concentrations were compared to δ13C of bulk material, soluble carbon extracts, and the neutral sugar fraction from leaves. Amino acid composition and inorganic ions of the phloem sap was also analysed. Quantitative, systematic changes were detected in phloem sap composition and δ13C in response to altered water availability. Phloem sap δ13C was more sensitive to changes of water availability than the δ13C of bulk leaf, the soluble carbon fraction, and the neutral soluble fraction of leaves. Changes in water availability also resulted in significant changes in phloem sugar (sucrose and raffinose), inorganic nutrient (potassium), and amino acid (phenylalanine) concentrations with important implications for the maintenance of phloem function and biomass partitioning. The differences in carbohydrate and amino acid composition as well as the δ13C in the phloem, along with a new model system for phloem research, offer an improved understanding of the phloem-mediated signal, nutrient, and photoassimilate transduction in relation to water availability.

Stable osmotica in Eucalyptus spathulata — responses to salt and water deficit stress

Salt and water deficit stress elicited contrasting responses in seedlings of Eucalyptus spathulata (Hook.). Under salt stress, seedlings reduced osmotic potential by accumulating large quantities of inorganic ions in leaf tissues. Sodium concentrations reached 350 mm on a leaf water basis and total cellular osmolality reached ∼2000 mm. Under water deficit stress, maximum sodium concentrations were around 50 mm in leaf water and seedlings reduced osmotic potential through increasing concentrations of a range of constitutive solutes up to a total cellular osmolality of ∼1200 mm. We postulate that measured concentrations of the cyclic polyol, quercitol, of up to 200 mm leaf water, are the likely means of balancing accumulation of inorganic ions. Under the common assumption of localisation of inorganic ions to the vacuole and organic balancing solutes to the cytoplasm, the concentrations of cyclitol, and other carbohydrates were more than sufficient to balance osmotic potential across the tonoplast membrane. Results confirm other recent studies that suggest a range of putative roles for cyclitols in tissues and these are discussed in the larger context of plant responses to salt and water deficit stress.

Compound‐specific differences in 13C of soluble carbohydrates in leaves and phloem of 6‐month‐old Eucalyptus globulus (Labill)

Movement of photoassimilates from leaves to phloem is an important step for the flux of carbon through plants. Fractionation of carbon isotopes during this process may influence their abundance in heterotrophic tissues. We subjected Eucalyptus globulus to 20, 25 and 28 °C ambient growth temperatures and measured compound-specific δ(13)C of carbohydrates obtained from leaves and bled phloem sap. We compared δ(13)C of sucrose and raffinose obtained from leaf or phloem and of total leaf soluble carbon, with modelled values predicted by leaf gas exchange. Changes in δ(13)C of sucrose and raffinose obtained from either leaves or phloem sap were more tightly coupled to changes in c(i)/c(a) than was δ(13)C of leaf soluble carbon. At 25 and 28 °C, sucrose and raffinose were enriched in (13)C compared to leaf soluble carbon and predicted values - irrespective of tissue type. Phloem sucrose was depleted and raffinose enriched in (13)C compared to leaf extracts. Intermolecular and tissue-specific δ(13)C reveal that multiple systematic factors influence (13)C composition during export to phloem. Predicting sensitivity of these factors to changes in plant physiological status will improve our ability to infer plant function at a range of temporal and spatial scales.

Relations of sugar composition and δ13C in phloem sap to growth and physiological performance of Eucalyptus globulus (Labill)

We characterized differences in carbon isotopic content (delta(13)C) and sugar concentrations in phloem exudates from Eucalyptus globulus (Labill) plantations across a rainfall gradient in south-western Australia. Phloem sap delta(13)C and sugar concentrations varied with season and annual rainfall. Annual bole growth was negatively related to phloem sap delta(13)C during summer, suggesting a water limitation, yet was positively related in winter. We conclude that when water is abundant, variations in carboxylation rates become significant to overall growth. Concentrations of sucrose in phloem sap varied across sites by up to 600 mm, and raffinose by 300 mm. These compounds play significant roles in maintaining osmotic balance and facilitating carbon movement into the phloem, and their relative abundances contribute strongly to overall delta(13)C of phloem sap. Taken together, the delta(13)C and concentrations of specific sugars in phloem sap provide significant insights to functions supporting growth at the tree, site and landscape scale.

Edge type affects leaf-level water relations and estimated transpiration of Eucalyptus arenacea.

While edge effects on tree water relations are well described for closed forests, they remain under-examined in more open forest types. Similarly, there has been minimal evaluation of the effects of contrasting land uses on the water relations of open forest types in highly fragmented landscapes. We examined edge effects on the water relations and gas exchange of a dominant tree (Eucalyptus arenacea Marginson & Ladiges) in an open forest type (temperate woodland) of south-eastern Australia. Edge effects in replicate woodlands adjoined by cleared agricultural land (pasture edges) were compared with those adjoined by 7- to 9-year-old eucalypt plantation with a 25m fire break (plantation edges). Consistent with studies in closed forest types, edge effects were pronounced at pasture edges where photosynthesis, transpiration and stomatal conductance were greater for edge trees than interior trees (75m into woodlands), and were related to greater light availability and significantly higher branch water potentials at woodland edges than interiors. Nonetheless, gas exchange values were only ∼50% greater for edge than interior trees, compared with ∼200% previously found in closed forest types. In contrast to woodlands adjoined by pasture, gas exchange in winter was significantly lower for edge than interior trees in woodlands adjoined by plantations, consistent with shading and buffering effects of plantations on edge microclimate. Plantation edge effects were less pronounced in summer, although higher water use efficiency of edge than interior woodland trees indicated possible competition for water between plantation trees and woodland edge trees in the drier months (an effect that might have been more pronounced were there no firebreak between the two land uses). Scaling up of leaf-level water relations to stand transpiration using a Jarvis-type phenomenological model indicated similar differences between edge types. That is, transpiration was greater at pasture than plantation edges in summer months (most likely due to greater water availability at pasture edges), resulting in significantly greater estimates of annual transpiration at pasture than plantation edges (430 vs. 343lm(-2)year(-1), respectively). Our study highlights the need for landscape-level water flux models to account for edge effects on stand transpiration, particularly in highly fragmented landscapes.

Differences in ascorbate and glutathione levels as indicators of resistance and susceptibility in Eucalyptus trees infected with Phytophthora cinnamomi

In this study we investigated the role that ascorbate (AA) and glutathione (GSH) play in the plant pathogen interaction of susceptible Eucalyptus sieberi L. A. Johnson and resistant Eucalyptus sideroxylon Woolls with Phytophthora cinnamomi Rands root infection. In a glasshouse study, seedlings were grown in soil-free plant boxes to facilitate the inoculation of the root systems by a P. cinnamomi zoospore solution. Ascorbate and GSH concentrations were measured in infected roots and leaves, along with leaf gas exchange, chlorophyll fluorescence and carbohydrate concentrations over a time course up to 312 h (13 days) post-inoculation (pi). At the early stages of infection (from 24 h pi), significant decreases in AA and GSH concentrations were observed in the infected roots and leaves of the susceptible E. sieberi seedlings. At the later stage of infection (312 h pi), the earlier AA decreases in the leaves of infected plants had become significant increases. In contrast, late, significant AA increases in the absence of any GSH changes were observed in the infected roots of the resistant E. sideroxylon seedlings. In E. sideroxylon leaves, a significant GSH increase occurred at 24 h pi; however, by 312 h pi the earlier increase had become a significant decrease, while no changes occurred in AA. In E. sieberi, photosynthesis (A), stomatal conductance (g(s)) and PSII quantum efficiency (Φ(PSII)) were reduced by ~60, 80 and 30%, respectively, in infected plants and remained significantly lower than uninfected controls for the duration of the experiment. Significant reductions in these parameters did not occur until later (120 h pi for g(s) and 312 h pi for A and Φ(PSII)), and to a lesser extent in the resistant species. Non-structural carbohydrate analysis of roots and leaves indicate that carbohydrate metabolism and resource flow between shoots and roots may have been altered at later infection stages. This study suggests that reduced antioxidant capacity, leaf physiological function and carbohydrate metabolism are associated with susceptibility in E. sieberi to P. cinnamomi infection, while AA increases and new root formation were associated with resistance in E. sideroxylon.

Shade does not ameliorate drought effects on the tree fern species Dicksonia antarctica and Cyathea australis

We examined the responses of two tree fern species (Dicksonia antarctica and Cyathea australis) growing under moderate and high light regimes to short-term water deficit followed by rewatering. Under adequate water supply, morphological and photosynthetic characteristics differed between species. D. antarctica, although putatively the more shade and less drought adapted species, had greater chlorophyll a/b ratio, and greater water use efficiency and less negative δ13C. Both species were susceptible to water deficit regardless of the light regime showing significant decreases in photosynthetic parameters (Amax, Vcmax, Jmax) and stomatal conductance (gs) in conjunction with decreased relative frond water content (RWC) and predawn frond water potential (Ψpredawn). During the water deficit period, decreases in gs in both species started one day later, and were at lower soil water content, under moderate light compared with high light. D. antarctica under moderate light was more vulnerable to drought than all other plants as was indicated by greater decreases in Ψpredawn, lowest stomatal conductance, and photosynthetic rates. Both tree fern species were able to recover after a short but severe water stress.

Seasonal changes in carbohydrates, cyclitols, and water relations of 3 field grown Eucalyptus species from contrasting taxonomy on a common site

Abstract• Alterations in plant chemistry underpin a suite of physiological adaptations to arid conditions. Qualitative and quantitative differences in leaf chemistry are found in the genus Eucalyptus correlating with physiological adaptation to aridity.• Here we investigate seasonal water relations of three field grown eucalypt species grown at a common site known to differ in their ability to accumulate the cyclic sugar alcohol, quercitol.• We show that quercitol contributes significantly to osmotic relations in field grown trees of Eucalyptus melliodora but is present only in trace amounts in E. rubida and E. obliqua.• Measured concentrations of quercitol account for the difference in osmotic potentials between species and can be interpreted as a mechanism for adaptation to low water availability.Résumé• Des altérations dans la chimie des plants soutiennent une série d’adaptations physiologiques à l’aridité. Des différences qualitatives et quantitatives dans la chimie des feuilles sont trouvées chez le genre Eucalyptus en corrélation avec les adaptations physiologiques à l’aridité.• Ici, nous étudions les relations hydriques saisonnières de 3 espèces d’Eucalyptus de plein champ, poussant sur un site commun, connues pour être différentes au plan de leur capacité à accumuler le quercitol.• Nous montrons que le quercitol contribue de manière significative aux relations osmotiques chez Eucalyptus melliodora mais est seulement présent à l’état de trace chez Eucalyptus rubida et Eucalyptus obliqua.• Les concentrations de quercitol qui ont été mesurées rendent compte des différences de potentiel osmotique observées entre les espèces et peuvent être interprétées comme un mécanisme d’adaptation à la faible disponibilité en eau.

The Regulation of Osmotic Potential in Trees

The availability of water and the ability of plants to acquire it influence productivity among many ecosystems. For all plants, both terrestrial and marine, the modification of internal osmotic potential (ψπ) is a commonly observed response to changes in water availability. This chapter highlights that modification of ψπ and its effects on plant water potential (ψ) should be considered with regard to physiological relevance, particularly when discussing the physiology of trees. A limited range of solutes are suitable as cellular osmotica and we highlight the physiochemical properties that help to maintain physiological function at low ψ. Overall, differences in the capacity to regulate ψπ are among many functional adaptations that enable growth and productivity of trees across a wide range of environments.