Genetic Diversity in Nitrogen Fertiliser Responses and N Gas Emission in Modern Wheat.

Crops assimilate nitrogen (N) as ammonium via the glutamine synthetase/glutamate synthase (GS/GOGAT) pathway which is of central importance for N uptake and potentially represents a bottle neck for N fertiliser-use efficiency. The aim of this study was to assess whether genetic diversity for N-assimilation capacity exists in wheat and could be exploited for breeding. Wheat plants rapidly, within 6 h, responded to N application with an increase in GS activity. This was not accompanied by an increase in GS gene transcript abundance and a comparison of GS1 and GS2 protein models revealed a high degree of sequence conservation. N responsiveness amongst ten wheat varieties was assessed by measuring GS enzyme activity, leaf tissue ammonium, and by a leaf-disc assay as a proxy for apoplastic ammonia. Based on these data, a high-GS group showing an overall positive response to N could be distinguished from an inefficient, low-GS group. Subsequent gas emission measurements confirmed plant ammonia emission in response to N application and also revealed emission of N2O when N was provided as nitrate, which is in agreement with our current understanding that N2O is a by-product of nitrate reduction. Taken together, the data suggest that there is scope for improving N assimilation capacity in wheat and that further investigations into the regulation and role of GS-GOGAT in NH3 emission is justified. Likewise, emission of the climate gas N2O needs to be reduced, and future research should focus on assessing the nitrate reductase pathway in wheat and explore fertiliser management options.

Sugar sensing responses to low and high light in leaves of the C4 model grass Setaria viridis.

Although sugar regulates photosynthesis, the signalling pathways underlying this process remain elusive, especially for C4 crops. To address this knowledge gap and identify potential candidate genes, we treated Setaria viridis (C4 model) plants acclimated to medium light intensity (ML, 500 µmol m-2 s-1) with low (LL, 50 µmol m-2 s-1) or high (HL, 1000 µmol m-2 s-1) light for 4 d and observed the consequences on carbon metabolism and the transcriptome of source leaves. LL impaired photosynthesis and reduced leaf content of signalling sugars (glucose, sucrose, and trehalose-6-phosphate). In contrast, HL strongly induced sugar accumulation without repressing photosynthesis. LL more profoundly impacted the leaf transcriptome, including photosynthetic genes. LL and HL contrastingly altered the expression of hexokinase (HXK) and sucrose-non-fermenting 1 (Snf1)-related protein kinase 1 (SnRK1) sugar sensors and trehalose pathway genes. The expression of key target genes of HXK and SnRK1 were affected by LL and sugar depletion, while surprisingly HL and strong sugar accumulation only slightly repressed the SnRK1 signalling pathway. In conclusion, we demonstrate that LL profoundly impacted photosynthesis and the transcriptome of S. viridis source leaves, while HL altered sugar levels more than LL. We also present the first evidence that sugar signalling pathways in C4 source leaves may respond to light intensity and sugar accumulation differently from C3 source leaves.

Current and possible approaches for improving photosynthetic efficiency.

One of the most important tasks laying ahead today's biotechnology is to improve crop productivity with the aim of meeting increased food and energy demands of humankind. Plant productivity depends on many genetic factors, including life cycle, harvest index, stress tolerance and photosynthetic activity. Many approaches were already tested or suggested to improve either. Limitations of photosynthesis have also been uncovered and efforts been taken to increase its efficiency. Examples include decreasing photosynthetic antennae size, increasing the photosynthetically available light spectrum, countering oxygenase activity of Rubisco by implementing C4 photosynthesis to C3 plants and altering source to sink transport of metabolites. A natural and effective photosynthetic adaptation, the sugar alcohol metabolism got however remarkably little attention in the last years, despite being comparably efficient as C4, and can be considered easier to introduce to new species. We also propose root to shoot carbon-dioxide transport as a means to improve photosynthetic performance and drought tolerance at the same time. Different suggestions and successful examples are covered here for improving plant photosynthesis as well as novel perspectives are presented for future research.

The Arabidopsis thaliana N-recognin E3 ligase PROTEOLYSIS1 influences the immune response.

N-degron pathways of ubiquitin-mediated proteolysis (formerly known as the N-end rule pathway) control the stability of substrate proteins dependent on the amino-terminal (Nt) residue. Unlike yeast or mammalian N-recognin E3 ligases, which each recognize several different classes of Nt residues, in Arabidopsis thaliana, N-recognin functions of different N-degron pathways are carried out independently by PROTEOLYSIS (PRT)1, PRT6, and other unknown proteins. PRT1 recognizes type 2 aromatic Nt-destabilizing residues and PRT6 recognizes type 1 basic residues. These two N-recognin functions diverged as separate proteins early in the evolution of plants, before the conquest of the land. We demonstrate that loss of PRT1 function promotes the plant immune system, as mutant prt1-1 plants showed greater apoplastic resistance than WT to infection by the bacterial hemi-biotroph Pseudomonas syringae pv tomato (Pst) DC3000. Quantitative proteomics revealed increased accumulation of proteins associated with specific components of plant defense in the prt1-1 mutant, concomitant with increased accumulation of salicylic acid. The effects of the prt1 mutation were additional to known effects of prt6 in influencing the immune system, in particular, an observed over-accumulation of pipecolic acid (Pip) in the double-mutant prt1-1 prt6-1. These results demonstrate a potential role for PRT1 in controlling aspects of the plant immune system and suggest that PRT1 limits the onset of the defense response via degradation of substrates with type 2 Nt-destabilizing residues.

Trehalose 6-Phosphate Regulates Photosynthesis and Assimilate Partitioning in Reproductive Tissue.

Transgenic maize (Zea mays) that expresses rice (Oryza sativa) TREHALOSE PHOSPHATE PHOSPHATASE1 (TPP1) from the rice MADS6 promoter, which is active over the flowering period, produces higher yields than wild type. This yield increase occurs with or without drought conditions during flowering. To understand the mechanistic basis of the increased yield, we characterized gene expression and metabolite profiles in leaves and developing female reproductive tissue, comprising florets, node, pith, and shank, over the flowering period with and without drought. The MADS6 promoter was most active in the vasculature, particularly phloem companion cells in florets and pith, consistent with the largest decreases in trehalose 6-phosphate (T6P) levels (2- to 3-fold) being found in pith and florets. Low T6P led to decreased gene expression for primary metabolism and increased gene expression for secondary metabolism, particularly lipid-related pathways. Despite similar changes in gene expression, the pith and floret displayed opposing assimilate profiles: sugars, sugar phosphates, amino acids, and lipids increased in florets, but decreased in pith. Possibly explaining this assimilate distribution, seven SWEET genes were found to be up-regulated in the transgenic plants. SnRK1 activity and the expression of the gene for the SnRK1 beta subunit, expression of SnRK1 marker genes, and endogenous trehalose pathway genes were also altered. Furthermore, leaves of the transgenic maize maintained a higher photosynthetic rate for a longer period compared to wild type. In conclusion, we found that decreasing T6P in reproductive tissues down-regulates primary metabolism and up-regulates secondary metabolism, resulting in different metabolite profiles in component tissues. Our data implicate T6P/ SnRK1 as a major regulator of whole-plant resource allocation for crop yield improvement.

Increasing crop yield and resilience with trehalose 6-phosphate: targeting a feast-famine mechanism in cereals for better source-sink optimization.

Food security is a pressing global issue. New approaches are required to break through a yield ceiling that has developed in recent years for the major crops. As important as increasing yield potential is the protection of yield from abiotic stresses in an increasingly variable and unpredictable climate. Current strategies to improve yield include conventional breeding, marker-assisted breeding, quantitative trait loci (QTLs), mutagenesis, creation of hybrids, genetic modification (GM), emerging genome-editing technologies, and chemical approaches. A regulatory mechanism amenable to three of these approaches has great promise for large yield improvements. Trehalose 6-phosphate (T6P) synthesized in the low-flux trehalose biosynthetic pathway signals the availability of sucrose in plant cells as part of a whole-plant sucrose homeostatic mechanism. Modifying T6P content by GM, marker-assisted selection, and novel chemistry has improved yield in three major cereals under a range of water availabilities from severe drought through to flooding. Yield improvements have been achieved by altering carbon allocation and how carbon is used. Targeting T6P both temporally and spatially offers great promise for large yield improvements in productive (up to 20%) and marginal environments (up to 120%). This opinion paper highlights this important breakthrough in fundamental science for crop improvement.

Plastid Molecular Pharming I. Production of Oral Vaccines via Plastid Transformation.

BACKGROUND: Vaccines produced in plants have opened up new opportunities in vaccination. OBJECTIVE: Among the various categories of vaccines, the recombinant vaccine is generally regarded as the most economical and safest type because it cannot cause disease and does not require large-scale cultivation of pathogens. Due to the low cost of their cultivation, plants may represent viable alternative platforms for producing subunit vaccines. Genetic engineering of plastids is the innovation of the last three decades and has numerous benefits when compared to nuclear transformation. Due to the high level of expression, oral vaccines produced in transplastomic plants do not have to be purified as they can be consumed raw, which, therefore, reduces the cost of preparation, transportation and handling of the vaccines. Oral vaccination also excludes the risk of other infections or contaminations, while compartmentation of the plant cell provides an excellent encapsulation to the antigen within the plastid. RESULTS & CONCLUSION: Herein we review the main biotechnological and immunological aspects of the progress achieved in the field of plastid derived edible vaccines during the last decade. As there is a public debate against genetically modified crops, the advantages and limitations of oral vaccines are also discussed.

The trafficking pathway of a wheat storage protein in transgenic rice endosperm.

BACKGROUND AND AIMS: The trafficking of proteins in the endoplasmic reticulum (ER) of plant cells is a topic of considerable interest since this organelle serves as an entry point for proteins destined for other organelles, as well as for the ER itself. In the current work, transgenic rice was used to study the pattern and pathway of deposition of the wheat high molecular weight (HMW) glutenin sub-unit (GS) 1Dx5 within the rice endosperm using specific antibodies to determine whether it is deposited in the same or different protein bodies from the rice storage proteins, and whether it is located in the same or separate phases within these. METHODS: The protein distribution and the expression pattern of HMW sub-unit 1Dx5 in transgenic rice endosperm at different stages of development were determined using light and electron microscopy after labelling with antibodies. KEY RESULTS: The use of HMW-GS-specific antibodies showed that sub-unit 1Dx5 was expressed mainly in the sub-aleurone cells of the endosperm and that it was deposited in both types of protein body present in the rice endosperm: derived from the ER and containing prolamins, and derived from the vacuole and containing glutelins. In addition, new types of protein bodies were also formed within the endosperm cells. CONCLUSIONS: The results suggest that the HMW 1Dx5 protein could be trafficked by either the ER or vacuolar pathway, possibly depending on the stage of development, and that its accumulation in the rice endosperm could compromise the structural integrity of protein bodies and their segregation into two distinct populations in the mature endosperm.

Wheat storage proteins in transgenic rice endosperm.

Transgenic rice seed expressing wheat HMW glutenin subunit was characterized to study the effects of the wheat prolamin on the protein expression pattern and protein size distribution in the endosperm and the functional and rheological properties of the rice flour and dough. Significant differences were found in the protein expression pattern between the transgenic and wild type samples. Comparing the protein expression profiles of transgenic and nontransgenic plants, combined with proteomic-based studies, indicated increased protein disulfide isomerase (PDI) levels in the transgenic rice lines. The accurate molecular size of HMW-GS in rice endosperm was identified by MALDI-TOF-MS analysis. The expressed wheat HMW (subunit 1Dx5) GS showed a positive effect on the functional properties of rice dough by significantly increasing the size distribution of the polymeric protein fraction and modifying the dough mixing parameters.

Comparative study of the effect of incorporated individual wheat storage proteins on mixing properties of rice and wheat doughs.

The aim of this work was to compare the effects of incorporated wheat storage proteins on the functional properties of rice and wheat flours. The advantage of rice as a base flour compared to wheat is that it does not contain any wheat flour components and, therefore, has no interactive effect between wheat glutenin proteins. The incorporation of individual HMW glutenin subunit proteins (Bx6, Bx7, and By8) in different ratios had significant positive effects on the mixing requirements of both rice and wheat doughs. Reconstitution experiments using two x+y type HMW-GS pairs together with a bacterially expressed LMW-GS have been also carried out in this study. The largest effects of polymer formation and mixing properties of rice flour dough were observed when Bx and By subunits were used in a 1:1 ratio and HMW and LMW glutenin subunits in a 1:3 ratio. However, using the same subunit ratios in wheat as the base flour, these synergistic effects were not observed.

Effects of wheat storage proteins on the functional properties of rice dough.

The objective of this work was to develop a rice flour based procedure for in vitro structure-function studies of wheat proteins. Rice flour has an advantage over wheat flour, because the signal/noise ratio should be higher after the incorporation of the wheat prolamins into the protein matrix of the dough. A reduction/oxidation procedure has been developed to incorporate glutenin subunits into the polymeric structure of rice dough protein. The results indicated that incorporation of bulk fractions of HMW and LMW glutenin subunits increased the mixing requirements of the dough, whereas simple addition resulted in weaker dough. The incorporation studies of individual HMW subunits (Bx6, Bx7, and By8) demonstrated that rice flour can be used to study and compare the functional properties of different glutenin subunits.

Expression of cholera toxin B subunit in transgenic rice endosperm.

The synthetic cholera toxin B subunit (CTB) gene, modified according to the optimized codon usage of plant genes, was introduced into a plant expression vector and expressed under the control of the Bx17 HMW (high molecular weight) wheat endosperm-specific promoter containing an intron of the rice act1. The recombinant vector was transformed into rice plants using a biolistic-mediated transformation method. Stable integration of the synthetic CTB gene into the chromosomal DNA was confirmed by PCR amplification analysis. A high level of CTB (2.1% of total soluble protein) was expressed in the endosperm tissue of the transgenic rice plants. The synthetic CTB produced only in the rice endosperm demonstrated strong affinity for G(M1)-ganglioside, thereby suggesting that the CTB subunits formed an active pentamer. The successful expression of CTB genes in transgenic plants makes it a powerful tool for the development of a plant-derived edible vaccine.

Expression of a synthetic neutralizing epitope of porcine epidemic diarrhea virus fused with synthetic B subunit of Escherichia coli heat labile enterotoxin in rice endosperm.

Epitopes often require co-delivery with adjuvant and targeting proteins to enable recognition by the immune system, and this approach may also increase the efficacy of the antigen. In this study, we assess and describe the ability of transgenic rice plants to express a fusion protein consisting of the B-subunit of the Escherichia coli heat-labile enterotoxin (LTB) and a synthetic core-neutralizing epitope (COE) of porcine epidemic diarrhea virus (PEDV), inducing an enteric disease that is seen most predominantly in piglets. Both components of the fusion proteins were detected with Western blot analysis. The fusion protein was determined to assemble into pentamers, as was evidenced by its ability to bind to GM1 gangliosides, and evidenced an average level of expression in a transgenic rice endosperm. This indicates that the expression system of the plant is capable of generating a sizable amount of antigen, possibly allowing for the successful development of an edible vaccine.