Zaxinone Synthase overexpression modulates rice physiology and metabolism, enhancing nutrient uptake, growth and productivity.

The rice Zaxinone Synthase (ZAS) gene encodes a carotenoid cleavage dioxygenase (CCD) that forms the apocarotenoid growth regulator zaxinone in vitro. Here, we generated and characterized constitutive ZAS-overexpressing rice lines, to better understand ZAS role in determining zaxinone content and regulating growth and architecture. ZAS overexpression enhanced endogenous zaxinone level, promoted root growth and increased the number of productive tillers, leading to about 30% higher grain yield per plant. Hormone analysis revealed a decrease in strigolactone (SL) content, which we confirmed by rescuing the high-tillering phenotype through application of a SL analogue. Metabolomics analysis revealed that ZAS overexpressing plants accumulate higher amounts of monosaccharide sugars, in line with transcriptome analysis. Moreover, transgenic plants showed higher carbon (C) assimilation rate and elevated root phosphate, nitrate and sulphate level, enhancing the tolerance towards low phosphate (Pi). Our study confirms ZAS as an important determinant of rice growth and architecture and shows that ZAS regulates hormone homoeostasis and a combination of physiological processes to promote growth and grain yield, which makes this gene an excellent candidate for sustainable crop improvement.

The intersection of nitrogen nutrition and water use in plants: new paths toward improved crop productivity.

Water and nitrogen availability limit crop productivity globally more than most other environmental factors. Plant availability of macronutrients such as nitrate is, to a large extent, regulated by the amount of water available in the soil, and, during drought episodes, crops can become simultaneously water and nitrogen limited. In this review, we explore the intricate relationship between water and nitrogen transport in plants, from transpiration-driven mass flow in the soil to uptake by roots via membrane transporters and channels and transport to aerial organs. We discuss the roles of root architecture and of suberized hydrophobic root barriers governing apoplastic water and nitrogen movement into the vascular system. We also highlight the need to identify the signalling cascades regulating water and nitrogen transport, as well as the need for targeted physiological analyses of plant traits influencing water and nitrogen uptake. We further advocate for incorporation of new phenotyping technologies, breeding strategies, and agronomic practices to improve crop yield in water- and nitrogen-limited production systems.

Opposite fates of the purine metabolite allantoin under water and nitrogen limitations in bread wheat.

KEY MESSAGE: Degradation of nitrogen-rich purines is tightly and oppositely regulated under drought and low nitrogen supply in bread wheat. Allantoin is a key target metabolite for improving nitrogen homeostasis under stress. The metabolite allantoin is an intermediate of the catabolism of purines (components of nucleotides) and is known for its housekeeping role in nitrogen (N) recycling and also for its function in N transport and storage in nodulated legumes. Allantoin was also shown to differentially accumulate upon abiotic stress in a range of plant species but little is known about its role in cereals. To address this, purine catabolic pathway genes were identified in hexaploid bread wheat and their chromosomal location was experimentally validated. A comparative study of two Australian bread wheat genotypes revealed a highly significant increase of allantoin (up to 29-fold) under drought. In contrast, allantoin significantly decreased (up to 22-fold) in response to N deficiency. The observed changes were accompanied by transcriptional adjustment of key purine catabolic genes, suggesting that the recycling of purine-derived N is tightly regulated under stress. We propose opposite fates of allantoin in plants under stress: the accumulation of allantoin under drought circumvents its degradation to ammonium (NH4+) thereby preventing N losses. On the other hand, under N deficiency, increasing the NH4+ liberated via allantoin catabolism contributes towards the maintenance of N homeostasis.

RootGraph: a graphic optimization tool for automated image analysis of plant roots.

This paper outlines a numerical scheme for accurate, detailed, and high-throughput image analysis of plant roots. In contrast to existing root image analysis tools that focus on root system-average traits, a novel, fully automated and robust approach for the detailed characterization of root traits, based on a graph optimization process is presented. The scheme, firstly, distinguishes primary roots from lateral roots and, secondly, quantifies a broad spectrum of root traits for each identified primary and lateral root. Thirdly, it associates lateral roots and their properties with the specific primary root from which the laterals emerge. The performance of this approach was evaluated through comparisons with other automated and semi-automated software solutions as well as against results based on manual measurements. The comparisons and subsequent application of the algorithm to an array of experimental data demonstrate that this method outperforms existing methods in terms of accuracy, robustness, and the ability to process root images under high-throughput conditions.

SOS1 tonoplast neo-localization and the RGG protein SALTY are important in the extreme salinity tolerance of Salicornia bigelovii.

The identification of genes involved in salinity tolerance has primarily focused on model plants and crops. However, plants naturally adapted to highly saline environments offer valuable insights into tolerance to extreme salinity. Salicornia plants grow in coastal salt marshes, stimulated by NaCl. To understand this tolerance, we generated genome sequences of two Salicornia species and analyzed the transcriptomic and proteomic responses of Salicornia bigelovii to NaCl. Subcellular membrane proteomes reveal that SbiSOS1, a homolog of the well-known SALT-OVERLY-SENSITIVE 1 (SOS1) protein, appears to localize to the tonoplast, consistent with subcellular localization assays in tobacco. This neo-localized protein can pump Na+ into the vacuole, preventing toxicity in the cytosol. We further identify 11 proteins of interest, of which SbiSALTY, substantially improves yeast growth on saline media. Structural characterization using NMR identified it as an intrinsically disordered protein, localizing to the endoplasmic reticulum in planta, where it can interact with ribosomes and RNA, stabilizing or protecting them during salt stress.

Salt-Tolerant Crops: Time to Deliver.

Despite the numerous advances made in our understanding of the physiology and molecular genetics of salinity tolerance, there have been relatively few applications of these to improve the salt tolerance of crops. The most significant advances have historically utilized intraspecific variation, introgression of traits from close crop wild relatives, or, less frequently, introgression from more distant relatives. Advanced lines often fail due to difficulties in the introgression or tracking of traits or due to yield penalties associated with the alleles in nonsaline environments. However, the greatest limitation is that salinity is not a primary trait for breeders. We must close the gap between research and delivery, especially for farmers who have precious few alternatives. These efforts should include a reassessment of old techniques such as grafting current crops with salt-tolerant hybrid rootstocks. Alternatively, future crops can be produced via domestication of salt-tolerant wild species-an approach that is now feasible in our lifetime.

Strategies for engineering improved nitrogen use efficiency in crop plants via redistribution and recycling of organic nitrogen.

Global use of nitrogen (N) fertilizers has increased sevenfold from 1960 to 1995 but much of the N applied is lost to the environment. Modifying the temporal and spatial distribution of organic N within the plant can lead to improved grain yield and/or grain protein content for the same or reduced N fertilizer inputs. Biotechnological approaches to modify whole plant distribution of amino acids and ureides has proven successful in several crop species. Manipulating selective autophagy pathways in crops has also improved N remobilization efficiency to sink tissues whilst the contribution of ribophagy, RNA and purine catabolism to N recycling in crops is still too early to foretell. Improved recycling and remobilization of N must exploit N-stress responsive transcriptional regulators, N-sensing or phloem-localized promotors and genetic variation for N-responsive traits.

Alterations in the mitochondrial alternative NAD(P)H Dehydrogenase NDB4 lead to changes in mitochondrial electron transport chain composition, plant growth and response to oxidative stress.

The branched respiratory electron transport chain of plants contains a non-phosphorylating alternative pathway consisting of type II NAD(P)H dehydrogenases on both sides of the inner membrane linked through the ubiquinone pool to an alternative oxidase (AOX). T-DNA and RNA interference (RNAi) were used to reduce gene expression to characterize the external NAD(P)H dehydrogenase NDB4 in Arabidopsis. The ndb4 lines showed different levels of suppression of NDB4 protein, leading to increases in NBD2 and AOX1a mRNA and protein levels in all lines. These changes were associated with lower reactive oxygen species formation and an altered phenotype, including changes in growth rate, root : shoot ratios and leaf area. The general growth pattern for the ndb4 mutants was decreased leaf area early in development (6-15 d) followed by a prompt subsequent increase in leaf area that exceeded the leaf area of the wild type by maturity (the 10-12 rosette stage). This pattern was most evident for the RNAi lines that had increased mitochondrial electron transport capacity. The RNAi lines also exhibited better tolerance to salinity stress, with better growth rates and lower shoot Na⁺ content compared with controls when grown under saline conditions. We hypothesize that these differences reflect the enhanced expression of NDB2 and AOX in the ndb4 mutant plants.

The role of light in the regulation of ascorbate metabolism during berry development in the cultivated grapevine Vitis vinifera L.

BACKGROUND: The accumulation of L-ascorbate (Asc) in fruits is influenced by environmental factors including light quantity. Fruit exposure to ambient light is often reduced by the surrounding leaf canopy, and can be altered by cultivation practices. The influence of reduced sunlight exposure on the accumulation of Asc and its catabolites was investigated in field-grown berries of the cultivated grapevine Vitis vinifera L. cv. Shiraz. RESULTS: Growth under sunlight-eliminated conditions resulted in reduced berry fresh weight, chlorosis and a reduced total L-ascorbate pool size. The concentration of the Asc catabolite L-tartaric acid (TA) was reduced in berries grown without light. Conversely, concentrations of oxalic acid (OA), an alternative catabolite of Asc, and malic acid (MA), were unaffected by shading the berries during development. Brief and significant reductions in transcription of the Asc metabolic genes were observed in shade-grown berries after 4 weeks of dark acclimatisation whilst a key TA biosynthetic gene was not regulated by light. CONCLUSIONS: The results demonstrate that light-regulation of Asc and TA occurs only at brief stages of development and that OA and MA accumulation is light independent. Additionally, the comparable ratios of TA product to Asc precursor under both light regimes suggest that the diversion of Asc to TA is driven by factors that are not responsive to light. These findings suggest that an altered light regime is not the key to manipulating TA or MA levels in the harvested berry.

The phosphoproteome of rice leaves responds to water and nitrogen supply.

The scarcity of freshwater is an increasing concern in flood-irrigated rice, whilst excessive use of nitrogen fertilizers is costly and contributes to environmental pollution. To co-ordinate growth adaptation under prolonged exposure to limited water or excess nitrogen supply, plants employ complex systems for signalling and regulation of metabolic processes. There is limited information on the involvement of one of the most important post-translational modifications (PTMs), protein phosphorylation, in plant adaptation to long-term changes in resource supply. Oryza sativa cv. Nipponbare was grown under two regimes of nitrogen from the time of germination to final harvest. Twenty-five days after germination, water was withheld from half the pots in each nitrogen treatment and low water supply continued for an additional 26 days, while the remaining pots were well watered. Leaves from all four groups of plants were harvested after 51 days in order to test whether phosphorylation of leaf proteins responded to prior abiotic stress events. The dominant impact of these resources is exerted in leaves, where PTMs have been predicted to occur. Proteins were extracted and phosphopeptides were analysed by nanoLC-MS/MS analysis, coupled with label-free quantitation. Water and nitrogen regimes triggered extensive changes in phosphorylation of proteins involved in membrane transport, such as the aquaporin OsPIP2-6, a water channel protein. Our study reveals phosphorylation of several peptides belonging to proteins involved in RNA-processing and carbohydrate metabolism, suggesting that phosphorylation events regulate the signalling cascades that are required to optimize plant response to resource supply.

Biosynthesis and Cellular Functions of Tartaric Acid in Grapevines.

Tartaric acid (TA) is an obscure end point to the catabolism of ascorbic acid (Asc). Here, it is proposed as a "specialized primary metabolite", originating from carbohydrate metabolism but with restricted distribution within the plant kingdom and lack of known function in primary metabolic pathways. Grapes fall into the list of high TA-accumulators, with biosynthesis occurring in both leaf and berry. Very little is known of the TA biosynthetic pathway enzymes in any plant species, although recently some progress has been made in this space. New technologies in grapevine research such as the development of global co-expression network analysis tools and genome-wide association studies, should enable more rapid progress. There is also a lack of information regarding roles for this organic acid in plant metabolism. Therefore this review aims to briefly summarize current knowledge about the key intermediates and enzymes of TA biosynthesis in grapes and the regulation of its precursor, ascorbate, followed by speculative discussion around the potential roles of TA based on current knowledge of Asc metabolism, TA biosynthetic enzymes and other aspects of fruit metabolism.

Quinoa Phenotyping Methodologies: An International Consensus.

Quinoa is a crop originating in the Andes but grown more widely and with the genetic potential for significant further expansion. Due to the phenotypic plasticity of quinoa, varieties need to be assessed across years and multiple locations. To improve comparability among field trials across the globe and to facilitate collaborations, components of the trials need to be kept consistent, including the type and methods of data collected. Here, an internationally open-access framework for phenotyping a wide range of quinoa features is proposed to facilitate the systematic agronomic, physiological and genetic characterization of quinoa for crop adaptation and improvement. Mature plant phenotyping is a central aspect of this paper, including detailed descriptions and the provision of phenotyping cards to facilitate consistency in data collection. High-throughput methods for multi-temporal phenotyping based on remote sensing technologies are described. Tools for higher-throughput post-harvest phenotyping of seeds are presented. A guideline for approaching quinoa field trials including the collection of environmental data and designing layouts with statistical robustness is suggested. To move towards developing resources for quinoa in line with major cereal crops, a database was created. The Quinoa Germinate Platform will serve as a central repository of data for quinoa researchers globally.

Ascorbate metabolism and the developmental demand for tartaric and oxalic acids in ripening grape berries.

BACKGROUND: Fresh fruits are well accepted as a good source of the dietary antioxidant ascorbic acid (Asc, Vitamin C). However, fruits such as grapes do not accumulate exceptionally high quantities of Asc. Grapes, unlike most other cultivated fruits do however use Asc as a precursor for the synthesis of both oxalic (OA) and tartaric acids (TA). TA is a commercially important product in the wine industry and due to its acidifying effect on crushed juice it can influence the organoleptic properties of the wine. Despite the interest in Asc accumulation in fruits, little is known about the mechanisms whereby Asc concentration is regulated. The purpose of this study was to gain insights into Asc metabolism in wine grapes (Vitis vinifera c.v. Shiraz.) and thus ascertain whether the developmental demand for TA and OA synthesis influences Asc accumulation in the berry. RESULTS: We provide evidence for developmentally differentiated up-regulation of Asc biosynthetic pathways and subsequent fluctuations in Asc, TA and OA accumulation. Rapid accumulation of Asc and a low Asc to dehydroascorbate (DHA) ratio in young berries was co-ordinated with up-regulation of three of the primary Asc biosynthetic (Smirnoff-Wheeler) pathway genes. Immature berries synthesised Asc in-situ from the primary pathway precursors D-mannose and L-galactose. Immature berries also accumulated TA in early berry development in co-ordination with up-regulation of a TA biosynthetic gene. In contrast, ripe berries have up-regulated expression of the alternative Asc biosynthetic pathway gene D-galacturonic acid reductase with only residual expression of Smirnoff-Wheeler Asc biosynthetic pathway genes and of the TA biosynthetic gene. The ripening phase was further associated with up-regulation of Asc recycling genes, a secondary phase of increased accumulation of Asc and an increase in the Asc to DHA ratio. CONCLUSION: We demonstrate strong developmental regulation of Asc biosynthetic, recycling and catabolic genes in grape berries. Integration of the transcript, radiotracer and metabolite data demonstrates that Asc and TA metabolism are developmentally regulated in grapevines; resulting in low accumulated levels of the biosynthetic intermediate Asc, and high accumulated levels of the metabolic end-product TA.

Manipulation of alternative oxidase can influence salt tolerance in Arabidopsis thaliana.

The growth and development of plants can be limited by environmental stresses such as salinity. It has been suggested that the non-phosphorylating alternative respiratory pathway in plants, mediated by the NAD(P)H dehydrogenase [NAD(P)H DH] and alternative oxidase (AOX), is important during environmental stresses. The involvement of this alternative pathway in a stress response may be linked to its capacity to uncouple carbon metabolism from adenylate control and/or the minimization of the formation of destructive reactive oxygen species (ROS). Salinity stress is a widespread, adverse environmental stress, which leads to an ionic imbalance, hyperosmotic stress and oxidative stress, the latter being the result of ROS formation. In this study, we show that salinity stress of Arabidopsis thaliana plants resulted in the formation of ROS, increased levels of Na+ in both the shoot and the root and an increase in transcription of Ataox1a, Atndb2 and Atndb4 genes, indicating the formation of an abridged non-phosphorylating electron transport chain in response to salinity stress. Furthermore, plants constitutively over-expressing Ataox1a, with increased AOX capacity, showed lower ROS formation, 30-40% improved growth rates and lower shoot Na+ content compared with controls, when grown under salinity stress conditions. Thus, more active AOX in roots and shoots can improve the salt tolerance of Arabidopsis as defined by its ability to grow more effectively in the presence of NaCl, and maintain lower shoot Na+ content. AOX does have an important role in stress adaptation in plants, and these results provide some validation of the hypothesis that AOX can play a critical role in cell re-programming under salinity stress.

RNA Catabolites Contribute to the Nitrogen Pool and Support Growth Recovery of Wheat.

Turn-over of RNA and catabolism of nucleotides releases one to four ammonia molecules; the released nutrients being reassimilated into primary metabolism. Preliminary evidence indicates that monocots store high levels of free nucleotides and nucleosides but their potential as a source of internal organic nitrogen for use and remobilization is uncharted. Early tillering wheat plants were therefore starved of N over a 5-day time-course with examination of nucleic acid yields in whole shoots, young and old leaves and roots. Nucleic acids constituted ∼4% of the total N pool of N starved wheat plants, which was comparable with the N available from nitrate (NO3 -) and greater than that available from the sum of 20 proteinogenic amino acids. Methods were optimized to detect nucleotide (purine and pyrimidine) metabolites, and wheat orthologs of RNA degradation (TaRNS), nucleoside transport (TaENT1, TaENT3) and salvage (TaADK) were identified. It was found that N starved wheat roots actively catabolised RNA and specific purines but accumulated pyrimidines. Reduced levels of RNA corresponded with induction of TaRNS2, TaENT1, TaENT3, and TaADK in the roots. Reduced levels of GMP, guanine, xanthine, allantoin, allantoate and glyoxylate in N starved roots correlated with accumulation of allantoate and glyoxylate in the oldest leaf, suggesting translocation of allantoin. Furthermore, N starved wheat plants exogenously supplied with N in the form of purine catabolites grew and photosynthesized as well as those plants re-supplied with NO3 -. These results support the hypothesis that the nitrogen and carbon recovered from purine metabolism can support wheat growth.

Genetic diversity for root plasticity and nitrogen uptake in wheat seedlings.

Enhancing nitrogen use efficiency (NUE) of wheat is a major focus for wheat breeding programs. NUE may be improved by identifying genotypes that are competitive for nitrogen (N) uptake in early vegetative stages of growth and are able to invest that N in grain. Breeders tend to select high yielding genotypes under conditions of medium to high N supply, but it is not known whether this influences the selection of root plasticity traits or whether, over time, breeders have selected genotypes with higher N uptake efficiency. To address this, genotypes were selected from CIMMYT (1966-1985) and Australian (1999-2007) breeding programs. Genotypes from both programs responded to low N supply by expanding their root surface area through increased total root number and/or length of lateral roots. Australian genotypes were N responsive (accumulated more N under high N than under low N) whereas CIMMYT genotypes were not very N responsive. This could not be explained by differences in N uptake capacity as shown by 15N flux analysis of two representative genotypes with contrasting N accumulation. Expression analysis of nitrate transporter genes revealed that the high-affinity transport system accounted for the majority of root nitrate uptake in wheat seedlings under both low and high N conditions.

Genome sequence of the Listia angolensis microsymbiont Microvirga lotononidis strain WSM3557(T.).

Microvirga lotononidis is a recently described species of root-nodule bacteria that is an effective nitrogen- (N2) fixing microsymbiont of the symbiotically specific African legume Listia angolensis (Welw. ex Bak.) B.-E. van Wyk & Boatwr. M. lotononidis possesses several properties that are unusual in root-nodule bacteria, including pigmentation and the ability to grow at temperatures of up to 45°C. Strain WSM3557(T) is an aerobic, motile, Gram-negative, non-spore-forming rod isolated from a L. angolensis root nodule collected in Chipata, Zambia in 1963. This is the first report of a complete genome sequence for the genus Microvirga. Here we describe the features of Microvirga lotononidis strain WSM3557(T), together with genome sequence information and annotation. The 7,082,538 high-quality-draft genome is arranged in 18 scaffolds of 104 contigs, contains 6,956 protein-coding genes and 84 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Program.

Genome sequence of the clover-nodulating Rhizobium leguminosarum bv. trifolii strain SRDI565.

Rhizobium leguminosarum bv. trifolii SRDI565 (syn. N8-J) is an aerobic, motile, Gram-negative, non-spore-forming rod. SRDI565 was isolated from a nodule recovered from the roots of the annual clover Trifolium subterraneum subsp. subterraneum grown in the greenhouse and inoculated with soil collected from New South Wales, Australia. SRDI565 has a broad host range for nodulation within the clover genus, however N2-fixation is sub-optimal with some Trifolium species and ineffective with others. Here we describe the features of R. leguminosarum bv. trifolii strain SRDI565, together with genome sequence information and annotation. The 6,905,599 bp high-quality-draft genome is arranged into 7 scaffolds of 7 contigs, contains 6,750 protein-coding genes and 86 RNA-only encoding genes, and is one of 100 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.