Abscisic acid-regulated protein degradation causes osmotic stress-induced accumulation of branched-chain amino acids in Arabidopsis thaliana.

MAIN CONCLUSION: Whereas proline accumulates through de novo biosynthesis in plants subjected to osmotic stress, leucine, isoleucine, and valine accumulation in drought-stressed Arabidopsis thaliana is caused by abscisic acid-regulated protein degradation. In response to several kinds of abiotic stress, plants greatly increase their accumulation of free amino acids. Although stress-induced proline increases have been studied the most extensively, the fold-increase of other amino acids, in particular branched-chain amino acids (BCAAs; leucine, isoleucine, and valine), is often higher than that of proline. In Arabidopsis thaliana (Arabidopsis), BCAAs accumulate in response to drought, salt, mannitol, polyethylene glycol, herbicide treatment, and nitrogen starvation. Plants that are deficient in abscisic acid signaling accumulate lower amounts of BCAAs, but not proline and most other amino acids. Previous bioinformatic studies had suggested that amino acid synthesis, rather than protein degradation, is responsible for the observed BCAA increase in osmotically stressed Arabidopsis. However, whereas treatment with the protease inhibitor MG132 decreased drought-induced BCAA accumulation, inhibition of BCAA biosynthesis with the acetolactate synthase inhibitors chlorsulfuron and imazapyr did not. Additionally, overexpression of BRANCHED-CHAIN AMINO ACID TRANSFERASE2 (BCAT2), which is upregulated in response to osmotic stress and functions in BCAA degradation, decreased drought-induced BCAA accumulation. Together, these results demonstrate that BCAA accumulation in osmotically stressed Arabidopsis is primarily the result of protein degradation. After relief of the osmotic stress, BCAA homeostasis is restored over time by amino acid degradation involving BCAT2. Thus, drought-induced BCAA accumulation is different from that of proline, which is accumulated due to de novo synthesis in an abscisic acid-independent manner and remains elevated for a more prolonged period of time after removal of the osmotic stress.

The catabolic enzyme methionine gamma-lyase limits methionine accumulation in potato tubers.

Increasing methionine in potato tubers is desirable, both to increase the availability of this limiting essential amino acid and to enhance the aroma of baked and fried potatoes. Previous attempts to elevate potato methionine content using transgenic approaches have focused on increasing methionine biosynthesis. Higher isoleucine accumulation in these transgenic tubers suggested that the potatoes compensate for increased methionine biosynthesis with enhanced catabolism via methionine gamma-lyase (MGL), thereby producing 2-ketybutyrate for isoleucine biosynthesis. In the current study, we show that potato StMGL1 encodes a functional MGL in potato tubers. In planta silencing of StMGL1 results in an increased methionine to isoleucine ratio in the free amino acid profile of potato tubers and, in some transgenic lines, elevated accumulation of free methionine. In both wild-type and transgenic tubers, the ratio of methionine to isoleucine is negatively correlated with the level of StMGL1 transcript. A three-dimensional distribution of free amino acids in potato tubers is also described.

Pleiotropic physiological consequences of feedback-insensitive phenylalanine biosynthesis in Arabidopsis thaliana.

A large proportion of plant carbon flow passes through the shikimate pathway to phenylalanine, which serves as a precursor for numerous secondary metabolites. To identify new regulatory mechanisms affecting phenylalanine metabolism, we isolated Arabidopsis thaliana mutants that are resistant to the phytotoxic amino acid m-tyrosine, a structural analog of phenylalanine. Map-based cloning identified adt2-1D, a dominant point mutation causing a predicted serine to alanine change in the regulatory domain of ADT2 (arogenate dehydratase 2). Relaxed feedback inhibition and increased expression of the mutant enzyme caused up to 160-fold higher accumulation of free phenylalanine in rosette leaves, as well as altered accumulation of several other primary and secondary metabolites. In particular, abundance of 2-phenylethylglucosinolate, which is normally almost undetectable in leaves of the A. thaliana Columbia-0 accession, is increased more than 30-fold. Other observed phenotypes of the adt2-1D mutant include abnormal leaf development, resistance to 5-methyltryptophan, reduced growth of the generalist lepidopteran herbivore Trichoplusia ni (cabbage looper) and increased salt tolerance.

GmDNJ1, a type-I heat shock protein 40 (HSP40), is responsible for both Growth and heat tolerance in soybean.

Global warming poses severe threats to agricultural production, including soybean. One of the major mechanisms for organisms to combat heat stress is through heat shock proteins (HSPs) that stabilize protein structures at above-optimum temperatures, by assisting in the folding of nascent, misfolded, or unfolded proteins. The HSP40 subgroups, or the J-domain proteins, functions as co-chaperones. They capture proteins that require folding or refolding and pass them on to HSP70 for processing. In this study, we have identified a type-I HSP40 gene in soybean, GmDNJ1, with high basal expression under normal growth conditions and also highly inducible under abiotic stresses, especially heat. Gmdnj1-knockout mutants had diminished growth in normal conditions, and when under heat stress, exhibited more severe browning, reduced chlorophyll contents, higher reactive oxygen species (ROS) contents, and higher induction of heat stress-responsive transcription factors and ROS-scavenging enzyme-encoding genes. Under both normal and heat-stress conditions, the mutant lines accumulated more aggregated proteins involved in protein catabolism, sugar metabolism, and membrane transportation, in both roots and leaves. In summary, GmDNJ1 plays crucial roles in the overall plant growth and heat tolerance in soybean, probably through the surveillance of misfolded proteins for refolding to maintain the full capacity of cellular functions.

A phased Vanilla planifolia genome enables genetic improvement of flavour and production.

The global supply of vanilla extract is primarily sourced from the cured beans of the tropical orchid species Vanilla planifolia. Vanilla plants were collected from Mesoamerica, clonally propagated and globally distributed as part of the early spice trade. Today, the global food and beverage industry depends on descendants of these original plants that have not generally benefited from genetic improvement. As a result, vanilla growers and processors struggle to meet global demand for vanilla extract and are challenged by inefficient and unsustainable production practices. Here, we report a chromosome-scale, phased V. planifolia genome, which reveals sequence variants for genes that may impact the vanillin pathway and therefore influence bean quality. Resequencing of related vanilla species, including the minor commercial species Vanilla × tahitensis, identified genes that could impact productivity and post-harvest losses through pod dehiscence, flower anatomy and disease resistance. The vanilla genome reported in this study may enable accelerated breeding of vanilla to improve high-value traits.

Reduced activity of Arabidopsis thaliana HMT2, a methionine biosynthetic enzyme, increases seed methionine content.

In the S-methylmethionine cycle of plants, homocysteine methyltransferase (HMT) catalyzes the formation of two molecules of methionine from homocysteine and S-methylmethionine, and methionine methyltransferase (MMT) catalyzes the formation of methionine from S-methylmethionine using S-adenosylmethionine as a methyl group donor. Somewhat surprisingly, two independently isolated knockdown mutations of HMT2 (At3g63250), one of three Arabidopsis thaliana genes encoding homocysteine methyltransferase, increased free methionine abundance in seeds. Crosses and flower stalk grafting experiments demonstrate that the maternal genotype at the top of the flower stalk determines the seed S-methylmethionine and methionine phenotype of hmt2 mutants. Uptake, transport and inter-conversion of [(13)C]S-methylmethionine and [(13)C]methionine in hmt2, mmt and wild-type plants show that S-methylmethionine is a non-essential intermediate in the movement of methionine from vegetative tissue to the seeds. Together, these results support a model whereby elevated S-methylmethionine in hmt2 vegetative tissue is transported to seeds and either directly or indirectly results in the biosynthesis of additional methionine. Manipulation of the S-methylmethionine cycle may provide a new approach for improving the nutritional value of major grain crops such as rice, as methionine is a limiting essential amino acid for mammalian diets.

meta-Tyrosine in Festuca rubra ssp. commutata (Chewings fescue) is synthesized by hydroxylation of phenylalanine.

m-Tyrosine is a non-protein amino acid that is structurally similar to the common protein amino acids p-tyrosine and phenylalanine. Copious amounts of m-tyrosine can be found in root exudates of the fine fescue cultivar, Festuca rubra L. ssp. commutata (Chewings fescue). The phytotoxicity of m-tyrosine may contribute to the allelopathic potential of F. rubra. m-Tyrosine in Euphorbia myrsinites (donkey-tail spurge), was previously shown to be synthesized via transamination of m-hydroxyphenylpyruvate. Here we show that m-tyrosine biosynthesis in F. rubra occurs through direct hydroxylation of phenylalanine in the root tips, perhaps through the activity of a cytochrome P450 enzyme. Hence, E. myrsinites and F. rubra, the only two plant species known to produce m-tyrosine, use distinct biosynthetic pathways that likely arose independently in evolutionary history.

Non-protein amino acids in plant defense against insect herbivores: representative cases and opportunities for further functional analysis.

Chemical defense against herbivores is of utmost importance for plants. Primary and secondary metabolites, including non-protein amino acids, have been implicated in plant defense against insect pests. High levels of non-protein amino acids have been identified in certain plant families, including legumes and grasses, where they have been associated with resistance to insect herbivory. Non-protein amino acids can have direct toxic effects via several mechanisms, including misincorporation into proteins, obstruction of primary metabolism, and mimicking and interfering with insect neurological processes. Additionally, certain non-protein amino acids allow nitrogen to be stored in a form that is metabolically inaccessible to herbivores and, in some cases, may act as signals for further plant defense responses. Specialized insect herbivores often possess specific mechanisms to avoid or detoxify non-protein amino acids from their host plants. Although hundreds of non-protein amino acids have been found in nature, biosynthetic pathways and defensive functions have been elucidated in only a few cases. Next-generation sequencing technologies and the development of additional plant and insect model species will facilitate further research on the production of non-protein amino acids, a widespread but relatively uninvestigated plant defense mechanism.

Grass roots chemistry: meta-tyrosine, an herbicidal nonprotein amino acid.

Fine fescue grasses displace neighboring plants by depositing large quantities of an aqueous phytotoxic root exudate in the soil rhizosphere. Via activity-guided fractionation, we have isolated and identified the nonprotein amino acid m-tyrosine as the major active component. m-Tyrosine is significantly more phytotoxic than its structural isomers o- and p-tyrosine. We show that m-tyrosine exposure results in growth inhibition for a wide range of plant species and propose that the release of this nonprotein amino acid interferes with root development of competing plants. Acid hydrolysis of total root protein from Arabidopsis thaliana showed incorporation of m-tyrosine, suggesting this as a possible mechanism of phytotoxicity. m-Tyrosine inhibition of A. thaliana root growth is counteracted by exogenous addition of protein amino acids, with phenylalanine having the most significant effect. The discovery of m-tyrosine, as well as a further understanding of its mode(s) of action, could lead to the development of biorational approaches to weed control.