Crop phenotyping in a context of global change: What to measure and how to do it.

High-throughput crop phenotyping, particularly under field conditions, is nowadays perceived as a key factor limiting crop genetic advance. Phenotyping not only facilitates conventional breeding, but it is necessary to fully exploit the capabilities of molecular breeding, and it can be exploited to predict breeding targets for the years ahead at the regional level through more advanced simulation models and decision support systems. In terms of phenotyping, it is necessary to determined which selection traits are relevant in each situation, and which phenotyping tools/methods are available to assess such traits. Remote sensing methodologies are currently the most popular approaches, even when lab-based analyses are still relevant in many circumstances. On top of that, data processing and automation, together with machine learning/deep learning are contributing to the wide range of applications for phenotyping. This review addresses spectral and red-green-blue sensing as the most popular remote sensing approaches, alongside stable isotope composition as an example of a lab-based tool, and root phenotyping, which represents one of the frontiers for field phenotyping. Further, we consider the two most promising forms of aerial platforms (unmanned aerial vehicle and satellites) and some of the emerging data-processing techniques. The review includes three Boxes that examine specific case studies.

Effect of irrigation salinity and ecotype on the growth, physiological indicators and seed yield and quality of Salicornia europaea.

The euhalophyte species Salicornia europaea is cultivated for oilseed and as a fodder crop in various parts of the world. In saline coastal environments it possesses great potential for the subsistence of the most disadvantaged farmers. We investigated the effect of salinity levels in irrigation water on the germination capacity, shoot biomass and seed productivity as well as diverse quality traits (nitrogen content in shoots and seeds and fatty acids, in seeds) and physiological traits (stable carbon and nitrogen isotopes and ion content) of two accessions collected in the United Arab Emirates (UAE). The three salinity levels tested were irrigation with fresh water (0.3 dS m-1), brackish water (25 dS m-1) and sea water (40 dS m-1). In addition, a hypersaline condition (80 dS m-1) was also tested for germination. The best germination rates were achieved with seeds exposed to fresh and brackish water, while imbibition with sea water decreased germination by half and hypersaline water inhibited it almost totally. However, the best irrigation regime in terms of biomass and seed yield involved brackish water. Moreover, rising salinity in the irrigation increased the stable isotope composition of carbon (δ13C) and nitrogen (δ15N), together with the Na+ and K+ of shoots and seeds, and the lipid levels of seeds, while the total nitrogen content and the profile of major fatty acids of seeds did not change. Differences between the two ecotypes existed for growth and seed yield with the best ecotype exhibiting lower δ13C and higher K+ in both shoots and seeds, lower Na+ and higher δ15N in shoots, and lower N in seeds, together with differences in major fatty acids. Physiological mechanisms behind the response to irrigation salinity and the ecotypic differences are discussed in terms of photosynthetic carbon and nitrogen metabolism.

The Hydrogen Isotope Composition δ2H Reflects Plant Performance.

The stable carbon (δ13C) and oxygen (δ18O) isotope compositions in plant matter reflect photosynthetic and transpirative conditions in plants, respectively. However, the nature of hydrogen isotope composition (δ2H) and what it reflects of plant performance is poorly understood. Using durum wheat (Triticum turgidum var durum), this study evaluated the effect of different water and nitrogen growing field conditions on transpiration and how this effect influenced the performance of δ2H in autotrophic (flag leaf), mixotrophic (ears), and heterotrophic (grains and roots) organs. Moreover, δ2H was compared with the δ13C and δ18O in the same organs. Isotope compositions were analyzed in dry matter, the water-soluble fraction, and in water from different tissues of a set of genotypes. Similar to δ13C, the δ2H correlated negatively with stomatal conductance, whereas no correlation was observed for δ18O. Moreover, δ2H was not only affected by changes in transpiration but also by photosynthetic reactions, probably as a consequence of NADPH formation in autotrophic organs. Compared with the δ2H of stem water, plant δ2H was strongly diminished in photosynthetic organs such as the flag leaves, whereas it strongly increased in heterotrophic organs such as grains and roots. In heterotrophic organs, δ2H was associated with postphotosynthetic effects because there are several processes that lead to 2H-enrichment of carbohydrates. In summary, δ2H exhibited specific features that inform about the water conditions of the wheat crop, together with the photosynthetic characteristics of the plant part considered. Moreover, correlations of δ2H with grain yield illustrate that this isotope can be used to assess plant performance under different growing conditions.

Identification of traits associated with barley yield performance using contrasting nitrogen fertilizations and genotypes.

Much attention has been paid to understanding the traits associated with crop performance and the associated underlying physiological mechanisms, with less effort done towards combining different plant scales, levels of observation, or including hybrids of autogamous species. We aim to identify mechanisms at canopy, leaf and transcript levels contributing to crop performance under contrasting nitrogen supplies in three barley genotypes, two hybrids and one commercial line. High nitrogen fertilization did not affect photosynthetic capacity on a leaf area basis and lowered nitrogen partial factor productivity past a certain point, but increased leaf area and biomass accumulation, parameters that were closely tracked using various different high throughput remote sensing based phenotyping techniques. These aspects, together with a larger catabolism of leaf nitrogen compounds amenable to sink translocation, contributed to higher crop production. Better crop yield and growth in hybrids compared to the line was linked to a nitrogen-saving strategy in source leaves to the detriment of larger sink size, as indicated by the lower leaf nitrogen content and downregulation of nitrogen metabolism and aquaporin genes. While these changes did not reduce photosynthesis capacity on an area basis, they were related with better nitrogen use in the hybrids compared with the line.

Contribution of the ear and the flag leaf to grain filling in durum wheat inferred from the carbon isotope signature: genotypic and growing conditions effects.

The ear, together with the flag leaf, is believed to play a major role as a source of assimilates during grain filling in C3 cereals. However, the intrusive nature of most of the available methodologies prevents reaching conclusive results in this regard. This study compares the carbon isotope composition (δ(13)C) in its natural abundance in the water-soluble fractions of the flag leaf blade and the ear with the δ(13)C of mature kernels to assess the relative contribution of both organs to grain filling in durum wheat (Triticum turgidum L. var. durum). The relative contribution of the ear was higher in landraces compared to modern cultivars, as well as in response to nitrogen fertilization and water stress. Such genotypic and environmentally driven differences were associated with changes in harvest index (HI), with the relative contribution of the ear being negatively associated with HI. In the case of the genotypic differences, the lower relative contribution of the ear in modern cultivars compared with landraces is probably associated with the appearance in the former of a certain amount of source limitation driven by a higher HI. In fact, the relative contribution of the ear was far more responsive to changes in HI in modern cultivars compared with landraces.

Physiological traits contributed to the recent increase in yield potential of winter wheat from Henan Province, China.

This experiment aims to test the traits responsible for the increase in yield potential of winter wheat released in Henan Province, China. Seven established cultivars released in the last 20 years and three advanced lines were assayed. The results showed that grain yield was positively correlated with harvest index (HI), kernel number per square meter, and aboveground biomass. In addition, the HI and aboveground biomass showed an increasing trend with the year of release. Therefore, we can conclude that bread wheat breeding advances during recent decades in Henan Province, China, have been achieved through an increase in HI, kernel number per square meter, and aboveground biomass. A higher δ(13)C seems also to be involved in these advances, which suggests a progressive improvement in constitutive water use efficiency not associated with a trend towards lower stomatal conductance in the most recent genotypes. However, genetic advance does not appear related to changes in photosynthesis rates on area basis when measured in the flag leaf or the spike, but only to a higher, whole-spike photosynthesis. Results also indirectly support the concept that under potential yield conditions, the spike contributed more than the flag leaf to kernel formation.

Molecular and physiological mechanisms associated with root exposure to mercury in barley.

Mercury (Hg) is a toxic metal that affects plant growth. Here the effect of Hg exposure on plant growth and leaf gas-exchange together with gene expression in roots is reported for barley. Hg was mainly accumulated in roots and only very small amounts were found in the shoots. Chlorophyll fluorescence and net photosynthesis were not affected by Hg. Nevertheless exposure to Hg reduced shoot and root growth, the shoot to root ratio, stomatal conductance, carbon isotope discrimination and expression of an aquaporin transcript, whereas abscisic acid related transcripts were over-expressed. These results suggested some degree of limitation to water uptake causing a moderate water stress when plants are exposed to Hg. Microarray (MapMan) analysis revealed changes in the transcription of genes involved in nitrogen metabolism, which were accompanied by decreased nitrogen concentrations in the shoots, together with an increase in transcripts associated with secondary metabolism, stress, inhibition of DNA synthesis/chromatin structure and cell organization elements. Moreover, Hg induced the expression of many transcripts known to be involved in the uptake, accumulation, transport and general responses to other heavy metals. It is concluded that barley is able to accumulate high amounts of Hg in roots through several transcriptional, metabolic and physiological adjustments.

Comparative performance of δ13C, δ18O and δ15N for phenotyping durum wheat adaptation to a dryland environment.

Grain yield and the natural abundance of the stable isotope compositions of carbon (δ13C), oxygen (δ18O) and nitrogen (δ15N) of mature kernels were measured during 3 consecutive years in 10 durum wheat genotypes (five landraces and five modern cultivars) subjected to different water and N availabilities in a Mediterranean location and encompassing a total of 12 trials. Water limitation was the main environmental factor affecting yield, δ13C and δ18O, whereas N fertilisation had a major effect on δ15N. The genotypic effect was significant for yield, yield components, δ13C, δ18O and δ15N. Landraces exhibited a higher δ13C and δ15N than cultivars. Phenotypic correlations of δ13C and δ18O with grain yield were negative, suggesting that genotypes able to sustain a higher water use and stomatal conductance were the most productive and best adapted; δ15N was also negatively correlated with grain yield regardless of the growing conditions. δ13C was the best isotopic trait in terms of genetic correlation with yield and heritability, whereas δ18O was the worst of the three isotopic abundances. The physiological basis for the different performance of the three isotopes explaining the genotypic variability in yield is discussed.

Ultrastructure and subcellular distribution of Cr in Iris pseudacorus L. using TEM and X-ray microanalysis.

Chromium pollution of freshwater is hazardous for humans and other organisms, and places a limitation on the use of polluted water sources. Phytoremediation, the use of plants to remove pollutants from the environment, is a cost-effective, environmentally friendly approach for water decontamination. To improve the efficiency of the process, it is essential to increase the current knowledge about Cr accumulation in macrophytes. Plants of Iris pseudacorus L. were treated with Cr(III) at 0.75 mM for 5 weeks to investigate Cr localization by means of transmission electron microscopy and energy dispersive X-ray analysis. Chromium induced severe ultrastructural alterations in the rhizodermis (cell wall disorganisation, thickening, plasmolysis, and electron-dense inclusions) and rhizome parenchyma (reduced cell size, cell wall detachment, vacuolation, and opaque granules). The highest Cr contents were found in the cell walls of the cortex in the roots and in the cytoplasm and intercellular spaces of the rhizome. The Cr concentration in root tissues was in the order cortex>rhizodermis>stele, whereas in the rhizome, Cr was evenly distributed. It is proposed that root and rhizome have distinct functions in the response of I. pseudacorus to Cr. The rhizodermis limits Cr uptake by means of Si deposition and cell wall thickening. The rhizome cortex generates vacuoles and granules where Cr co-occurs with S, indicating Cr sequestration by metal-binding proteins.

Quantitative trait loci for grain yield and adaptation of durum wheat (Triticum durum Desf.) across a wide range of water availability.

Grain yield is a major goal for the improvement of durum wheat, particularly in drought-prone areas. In this study, the genetic basis of grain yield (GY), heading date (HD), and plant height (PH) was investigated in a durum wheat population of 249 recombinant inbred lines evaluated in 16 environments (10 rainfed and 6 irrigated) characterized by a broad range of water availability and GY (from 5.6 to 58.8 q ha(-1)). Among the 16 quantitative trait loci (QTL) that affected GY, two major QTL on chromosomes 2BL and 3BS showed significant effects in 8 and 7 environments, with R2 values of 21.5 and 13.8% (mean data of all 16 environments), respectively. In both cases, extensive overlap was observed between the LOD profiles of GY and PH, but not with those for HD. QTL specific for PH were identified on chromosomes 1BS, 3AL, and 7AS. Additionally, three major QTL for HD on chromosomes 2AS, 2BL, and 7BS showed limited or no effects on GY. For both PH and GY, notable epistasis between the chromosome 2BL and 3BS QTL was detected across several environments.

Detection and quantification of unbound phytochelatin 2 in plant extracts of Brassica napus grown with different levels of mercury.

The mercury (Hg) accumulation mechanism was studied in rape (Brassica napus) plants grown under a Hg concentration gradient (0 microm-1,000 microm). Hg mainly accumulated in roots. Therefore, the presence of phytochelatins (PCs) was studied in the roots of the plants. The high stability of the PC-Hg multicomplexes (mPC-nHg) seems to be the main reason for the lack of previous Hg-PC characterization studies. We propose a modification of the method to detect and quantify unbound PC of Hg in plant extracts via high-performance liquid chromatography coupled to electrospray tandem mass spectrometry and inductively coupled plasma mass spectrometry in parallel. We separated the PC from the Hg by adding the chelating agent sodium 2,3-dimercaptopropanesulfonate monohydrate. We only detected the presence of PC after the addition of the chelating agent. Some multicomplexes mPC-nHg could be formed but, due to their large sizes, could not be detected. In this study, only PC(2) was observed in plant samples. Hg accumulation was correlated with PC(2) concentration (r(2) = 0.98).