The metabolic roles of free amino acids during seed development

Accepted Manuscript Title: The Metabolic Roles of Free Amino Acids during Seed Development Authors: Rachel Amir, Gad Galili, Hagai Cohen PII: DOI: Reference:

S0168-9452(18)30461-8 https://doi.org/10.1016/j.plantsci.2018.06.011 PSL 9880

To appear in:

Plant Science

Received date: Revised date: Accepted date:

25-4-2018 7-6-2018 13-6-2018

Please cite this article as: Amir R, Galili G, Cohen H, The Metabolic Roles of Free Amino Acids during Seed Development, Plant Science (2018), https://doi.org/10.1016/j.plantsci.2018.06.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The Metabolic Roles of Free Amino Acids during

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Seed Development

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Laboratory of Plant Science, Migal - Galilee Technology Center, Kiryat Shmona 12100,

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Israel; 2Tel-Hai College, Upper Galilee 11016, Israel; 3Department of Plant & Environmental

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Sciences, Weizmann Institute of Science, Rehovot 76100, Israel

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Rachel Amir1,2,* Gad Galili3 and Hagai Cohen3

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*Corresponding author: Rachel Amir

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Migal – Galilee Technology Center, P.O. Box 83, Kiryat Shmona 12100, Israel; email: [email protected]; Tel: 972-4-6953516; ORCID no. 0000-0003-0932-0724

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Highlights

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Amino acids play pivotal roles in seeds during their development

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The pools of free and total amino acids vary in different stages of seed development

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The biosynthetic pathways of amino acids in seeds are tightly regulated by metabolic

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networks

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Altered levels of amino acids can greatly affect seed physiology and behavior

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Abstract

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Amino acids play vital roles in the central metabolism of seeds. They are primarily utilized for

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the synthesis of seed-storage proteins, but also serve as precursors for the biosynthesis of

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accumulated in recent years describing the changes occurring in the contents of free amino

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acids (FAAs) during seed development. Since several essential amino acids are found in low

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levels in seeds (e.g., Lys, Met, Thr, Val, Leu, Ile and His), or play unique functional roles in

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seed development (e.g., Pro and the non-proteinogenic -aminobutyrate [GABA]), we also

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briefly describe studies carried out in order to alter their levels in seeds and determine the

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effects of the manipulation on seed biology. The lion share of these studies highlights strong

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positive correlations between the biosynthetic pathways of FAAs, meaning that when the levels

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of a certain amino acid change in seeds, the contents of other FAAs tend to elevate as well.

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These observations infer a tight regulatory network operating in the biosynthesis of FAAs

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secondary metabolites and as a source of energy. Here, we aimed at describing the knowledge

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during seed development.

compounds; genetic manipulation

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Keywords: amino acids; metabolic network; seed development; seed maturation; seed-storage

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Abbreviations: FAA, free amino acids; GABA, -aminobutyrate; TAA, total amino acids

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Table of content

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1. Introduction

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2. The Accumulation of Amino Acids during Seed Development

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3. The Various Effects of Altered Levels of Amino Acids on Seeds

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3.2 Met metabolism

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3.1 Lys metabolism

3.3 Thr metabolism

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3.4 Val, Ile and Leu metabolism 3.5 His metabolism

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3.6 Pro metabolism

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3.7 GABA metabolism

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4. Altered levels of certain amino acids can greatly impact the metabolism of other amino

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acids in seeds

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5. Conclusions and future prospective

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Plant seeds play significant roles in the plant life cycle as propagation units, but also contribute

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immensely to human and animal diets due to their high nutritional values contained in their

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protein, starch and oil contents. For these reasons, knowledge about seed biology could provide

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tools for managing genetic resources and improving agricultural products.

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1. Introduction

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embryo growth, cell division and morphogenesis; the second stage of seed maturation where

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The development of seeds can be divided into three fundamental stages: the first stage of

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entrance to dormancy [1-4]. These three stages are associated with substantial spatiotemporal

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metabolic rearrangements and major changes in global gene expression programs, protein

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profiles and metabolite levels [2, 5-7].

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massive accumulations of storage reserves occur; and the third stage of seed desiccation and

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developing seeds (e.g., [5, 8-10]). Despite their roles, the levels of FAAs in developing seeds

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are relatively much lower compared to the pool of total amino acids (TAAs), which include

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The pool of free amino acids (FAAs) plays key roles in the central metabolism of

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grains and Arabidopsis thaliana seeds, FAAs account for 1-10% and 7%, respectively, of

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those that are incorporated into seed-storage proteins. For example, in maize (Zea mays L.)

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FAA vary greatly between the three stages of seed development. According to Baud et al.

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TAAs [11, 12]. In terms of absolute quantity, several reports demonstrated that the levels of

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Gln, Gly and Ala, while at 11 DAF the levels of Leu and Val tend to increase dramatically.

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Then, at 16 DAF higher levels of Ser and Gly were detected [1]. As expected, due to different

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measurement techniques, growth conditions and exact definition of developmental stage, some

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other studies reported slightly different accumulation patterns of FAA during seed development

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[1, 2, 13, 14]. The levels of TAA were also measured in Arabidopsis seeds at 13, 17 and 28

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DAF, where the later stage represents fully-matured dry seeds [13]. According to this report,

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(2002), 6 days after flowering (DAF) the young Arabidopsis seed accumulates mostly Ser, Glu,

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weight, respectively. Additionally, Ala, Leu, Asp and Pro were accumulated to relatively high

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amounts, 10, 9, 8.5, and 8 mmol per g of dry weight) [13]. Markedly, the levels of the three

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branched-chain amino acids, Ile, Leu, Val, and those of Thr, were relatively constant in protein

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profiles of the Arabidopsis seed during development [13]. Together, these studies infer the

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complex regulation of FAAs biosynthesis and TAAs accumulation during the different stages

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of seed development.

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Gly and Glu are the predominant TAAs being accumulated up to 15 and 12 mmol per g of dry

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most important functional metabolic role in seeds, FAAs serve as one of the most important

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precursors for the synthesis of diverse groups of primary and secondary metabolites. These

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Even though the incorporation of FAAs into the seed-storage proteins is considered as its

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include organic acids, amino acids, osmolytes, phytohormones, and secondary metabolites

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which are integral components of the cell wall and thus involved in plant protection against

of accessible energy [9, 15, 16].

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various abiotic and biotic cues. In addition amino acids can be utilized as an alternative source

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This review aims at summarizing studies conducted in recent years that shed light on the

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studies that manipulated the levels of several amino acids, and their impacts on seed metabolic

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metabolism of FAAs during seed development. We also describe insights derived from recent

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profiles, global gene expression programs and other important seed traits.

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The functional regulatory roles of FAAs in developing seeds were investigated mainly in the

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Arabidopsis thaliana model plant. Generally, FAAs can be transported towards the seeds from

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vegetative tissues at the onset of reproductive tissues and seed development and/or through de

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novo synthesis occurring within the seed tissues. It was shown that during the first stage of seed

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2. The Accumulation of Amino Acids during Seed Development

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accompanied by the synthesis of reserve compounds in the developing seeds, particularly starch

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development, nutrients (mainly sugars) are taken up from the canopy. This process is

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fatty acids, which are further utilized for the synthesis of seed-storage proteins and oil,

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respectively [14, 17, 18]. These two types of storage compounds account for about 30-40% of

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the total dry matter of Arabidopsis seeds [1, 17, 19].

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[2, 4]. In the second stage of seed development, however, starch was converted into FAAs and

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stage of maturation and desiccation, the oil contents show opposite accumulation patterns and

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tend to decrease in these stages [1]. Correspondingly, the levels of several FAAs, degraded

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While the synthesis of seed-storage proteins continues during the third seed developmental

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fatty acids might support the boosted synthesis of FAAs at these stages, particularly those of

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fatty acids and sugars increase in the late stages [1]. It was suggested that the degradation of

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degradation [1]. In addition to Ser and Gly, the levels of aromatic amino acids Trp, Tyr and

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Ser and Gly, which can be synthesized by the glyoxylate pathway involved in fatty acid

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higher levels of secondary metabolites derived from these amino acids that are expected to play

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crucial roles in defense responses triggered naturally during seed germination [20, 21]. Similar

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trends were also reported in the levels of some nitrogen-rich amino acids, such as Asn, Arg,

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Lys and the non-proteinogenic amino acid GABA, suggesting that these specific FAAs are

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required during imbibition, the prior storage reserve degradation [5]. Among these, GABA is

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assumed to promote unceasing activity of the TCA cycle through the GABA shunt during early

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Phe tend to increase during the late stages [13, 20, 21]. These elevations were associated with

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was demonstrated not only by studies that utilized metabolite profiling approaches, but also by

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a proteomic study of mature dry seed of Lotus japonicus that inferred the enrichment of

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enzymes operating in the biosynthesis of amino acids [23]. These are also in agreement with

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findings showing high transcript expression levels of biosynthetic genes involved in the

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metabolism of amino acids at this stage [2]. Together, these observations suggest that even at

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later stages of seed development, some FAAs are still being synthesized de novo rather than

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being incorporated into seed-storage proteins.

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stages of germination [22]. The active ongoing biosynthesis of FAAs during seed desiccation

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during the development of Arabidopsis and Canola (Brassica napus) seeds, were able to show

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Despite these lines of evidence, other studies monitoring the levels of both FAAs and TAAs

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that while FAAs exhibit decreased amounts in this stage compared to other developmental

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stages, the levels of TAAs greatly increase [3, 13]. This implies that the majority of FAAs

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incorporate into seed-storage proteins during the late developmental stages [3, 13, 14]. Frank

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Arabidopsis seeds, demonstrating constant increased ratios of 15-, 75- and 275-fold at 13, 17

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et al. (2015) calculated the ratios of TAAs-to-FAAs at all three developmental stages of

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reduce during seed development to much higher extents compared to the levels of other FAAs,

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and 28 DAF, respectively. Among these FAA, the levels of Asn and Gln tend to significantly

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derived from their catabolic pathways [2].

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inferring their availability to synthesize seed-storage proteins as well as other amino acids

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additional studies highlighted the important contribution of transported FAAs synthesized in

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Apart from the de novo synthesis of FAAs within the seed tissues during their development,

vegetative tissues. The massive fluxes of FAAs towards the seeds are associated with leaves’

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senescence, i.e., during these processes, leaves degrade their proteins, which in turn result in a

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pool of FAAs available to transport towards the demanding developing seeds [24, 25]. In

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addition to FAA pool, it was demonstrated that during the onset of seed development as well

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crucial for the synthesis of some nitrogen-containing FAAs, that are later transferred into the

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developing seeds [27]. The ratio between the nitrogen taken up from the soil and nitrogen that

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is remobilized from the leaves, differ between plant species and depends on environmental and

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soil conditions [28]. In any case, amino acids are one of the main transported nitrogen-

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containing substances detected in the transport vessels of xylem and phloem [29]. For example,

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transported FAAs have been shown to account for 70-90% of total nitrogen content in rice

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grains [30]. Notably, Gln and Asn are the predominant amino acids found in xylem sap, while

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all other amino acids can be transported in phloem sap [29, 31], albeit at different rates and

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quantities. In rice, for example, it was found that Asp and Glu comprise 50% of total

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as its middle stages, nitrogen continues to be uptake by roots [26]. Its fixation in planta is

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transported FAAs [32], while in legumes such as pea (Pisum sativum L.), Lotus japonicus and

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soybean (Glycine Max L.), Asn was the predominant transported amino acid [33, 34]. In

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Brassica napus, however, Gln and Ala were the main ones [35]. These studies also

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proteins, but also for the synthesis of other amino acids through transamination/deamination

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reactions [33, 35].

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demonstrated that these transported FAAs are being utilized directly for the synthesis of

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The massive fluxes of FAAs from vegetative organs towards the developing seeds

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amino acid transporters have been identified in Arabidopsis [27, 36]. Given the complexity of

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amino acids transport and the high number of putative transporters in Arabidopsis, our

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understanding of the contribution of amino acids from non-seed tissues to the pool of FAAs

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necessitate specialized transporters capable of facilitating these processes. Over 100 putative

within seeds is still very partial [17, 29, 37]. An excellent example of such a dedicated amino

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acid transporter is the pea amino acid permease PsAAP1. Zhang et al. (2015) created transgenic

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seeds that overexpress this protein targeted to the sieve element-companion cell complexes of

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leaf phloem, as well as the epidermis of the seed cotyledons. As a result, transgenic plants were

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yield with improved total protein contents [34]. These observations alone clearly emphasize

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that our limited data regarding such dedicated transporters and the regulatory mechanisms by

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which they operate are delaying our understanding of the complete picture of how FAAs are

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being transported from vegetative organs towards developing seeds in plants.

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characterized by increased phloem and embryo loading of FAAs. The plants had higher seed

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3. The Various Effects of Altered Levels of Amino Acids on Seeds

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their major roles in seed metabolism and/or the fact that the levels of several amino acids limit

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the nutritional quality of seeds [9]. These nutritional-limiting amino acids belong to the group

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of essential amino acids, meaning that humans and animals cannot synthesize them de novo

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and need to consume them as part of their diets principally from plants [8, 9]. A large portion

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of knowledge regarding the functional roles of FAAs during seed development came from

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various studies that took the approach of genetic engineering and molecular manipulations to

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Naturally, amino acids attracted the attention of the scientific community principally due to

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consequences of these manipulations on seed biology were studied mainly by a battery of

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alter the levels of key genes operating in the biosynthetic pathways of several amino acids. The

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family pathway, Val, Ile, Leu from the branched-chain amino acids (BCAAs), His that belong

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diverse ‘omics’ analyses. In the current study we will refer to Lys, Met and Thr from the Asp-

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seed development. We briefly describe the predominant studies utilizing such tools that shed

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light on the metabolic roles of these important amino acids (Table 1, Fig. 1). The effects of

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these manipulations on seed biology and behavior are also discussed.

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to the essential amino acids, as well as Pro and GABA that play unique functional roles during

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Lys belongs to the Asp family of amino acids, and due to its relatively very low level in seeds

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(mainly cereal grains), researchers are investing efforts in elucidating its metabolic pathways

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and regulation [8, 9, 38]. To clarify the regulation of Lys and the possibilities of enhancing Lys

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production in Arabidopsis seeds, Zhu and Galili (2003, 2004) expressed, under the control of

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a seed-specific promoter, a bacterial feedback-insensitive dihydrodipicolinate synthase

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(DHDPS) enzyme of Lys synthesis in an Arabidopsis knockout mutant lacking a Lys catabolic

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enzyme of bifunctional Lys-ketoglutarate reductase/saccharopine dehydrogenase (LKR/SDH)

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enzyme. The seeds of these plants were shown to accumulate 80-fold more Lys content [39].

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Examining the effects of higher Lys content on the seed metabolic profile showed a significant

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3.1 Lys metabolism

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reduction in the levels of several tricarboxylic acid cycle (TCA) intermediates in both

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developing and germinating seeds. Yet, apart from the TCA cycle, no other metabolic

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perturbations were detected in these seeds [15, 40]. Despite these minor effects on the central

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programs stimulating the expression of hundreds of genes involved in anabolic processes

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associated with plant vigor, while suppressing a small number of genes participating in stress

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responses [40]. The most pronounced change was the induced expression of genes encoding

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ribosomal proteins, as well as those encoding translation, initiation and elongation factors, all

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metabolism of seeds, the higher levels of Lys had significant impacts on global gene expression

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lower germination capabilities apparently due to the lower levels of TCA cycle intermediates

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[15, 41]. Later, the authors were able to overcome these lower germination rates by generating

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of which are associated with protein synthesis [40]. As a result, the transgenic seeds displayed

transgenic Arabidopsis seeds that express the bacterial DHDPS enzyme together with the

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LKR/SDH-RNAi construct in a seed-specific manner, resulting in transgenic seeds with higher

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Lys levels but normal germination abilities [41].

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Met is synthesized via the Thr metabolic branch within the Asp-family pathway. Since the low

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levels of Met limit the nutritional quality of seeds, legume grains in particular, several

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manipulations were performed in order to increase its levels. The majority of work was done

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with different feedback-insensitive forms of the Arabidopsis cystathionine γ-synthase (CGS)

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[42], the main Met regulatory enzyme. Seeds produced by four different soybean (Glycine max)

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cultivars and Arabidopsis seeds expressing these mutated genes showed significantly higher

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levels of soluble Met [12, 43-45] . The expression of this gene in tobacco seeds did not yield

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higher soluble Met contents, but much higher total Met incorporated within the storage proteins

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of these transgenic seeds [12, 46]. The higher levels of soluble Met in these transgenic systems

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3.2 Met metabolism

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had an immense impact on other amino acids. In fact, higher levels of all FAAs were reported

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in these seeds, apart from Tyr [12, 47]. As a result, more FAAs were able to incorporate into

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seed-storage proteins, resulting in higher TAA and total protein contents [12, 24, 45].

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their seed-storage protein profiles, i.e., 12S-albumins and 2S-globulins, leading to higher

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quantities of some of their subunits. In turn, the higher total protein contents affected the total

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oil content of seeds, and altered the levels of some fatty acids, seed starch and water contents

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[48]. Apart from affecting the levels of storage compounds, the higher levels of Met in

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Transgenic Arabidopsis seeds with higher soluble Met contents had altered compositions of

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accumulated significantly higher levels of many stress-related primary metabolites such as

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soluble amino acids (Ile, Leu, Val and Pro), soluble sugars, TCA cycle intermediates and

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transgenic seeds had an immense impact on the central metabolome of seeds, which

polyamines. On the other hand, lower levels of GSH were measured in these seeds, apparently

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leading to their lower germination rates under oxidative stress conditions [12]. In agreement

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with these observations, the seeds’ transcriptome indicated a clear induction of dozens of

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stress-associated genes mostly those against osmotic and drought conditions, including those

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mediated by the signaling cascades of ethylene and abscisic acid (ABA). These changes

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implied stronger desiccation processes during the development of these seeds [12].

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be synthesized in seeds through an alternative pathway by which Met produced in vegetative

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tissues is converted to S-methyl-Met (SMM) that is transported towards the developing seeds.

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There, SMM is re-converted back to Met by methyltransferases called homocysteine

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methyltransferases (HMT). Arabidopsis hmt2 mutants lacking the activity of one of these

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methyltransferases exhibited 40-fold more soluble Met in its seeds compared to wild type (WT)

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seeds [49]. By producing transgenic Arabidopsis RNAi seeds with lower transcript expression

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of CGS, it was further revealed that SMM significantly contributes to the accumulation of Met

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Additional studies employing isotope-labeling techniques demonstrated that Met can also

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in seeds at late stages of seed development [24]. The levels of Met were also increased in seeds

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in plants overexpressing genes from the sulfur assimilation pathway. Overexpressing the

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bacterial serine acetyltransferase isoform (EcSAT) in rice (Oryza sativa L.) led to 1.4- and 4.8-

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in transgenic maize when this gene was employed, as well as by overexpressing the bacterial

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fold higher soluble and total Met in seeds, respectively [50]. Similar phenomena were observed

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sulfur assimilation pathway [16]. In both cases, the transgenic kernels had higher expression

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gene of 3'-phosphoadenosine-5'-phosphosulfate reductase (PAPR) that also operates in the

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these lines of evidence, another study showed that higher levels of Met and SMM in transgenic

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tobacco leaves did not affect the contents of Met in seeds [52], suggesting some complex

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regulatory metabolic networks in seeds that apparently can differ between tissues and/or

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of the Met-rich 10-kDa δ-zein, while the levels of other proteins did not change [51]. Despite

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species.

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Thr is one of several limiting essential amino acids of cereal grains [9]. Seed-specific

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expression of a bacterial feedback-insensitive form of Asp kinase in tobacco, narbon bean and

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soybean resulted in significantly higher Thr levels in seeds [53-55]. In tobacco seeds the level

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increased up to 16-fold in addition to 3-fold higher Met level compare to WT seeds [53]. These

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accumulations were accompanied by substantial increases in other major FAAs levels,

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resulting in up to a 3.5-fold increase in total FAA content in soybean [55]. In the latter

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transgenic seeds, the levels of Thr, Lys, Met, Ile and Trp accounted for more than 25% of total

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FAAs. Additionally, the levels of Ser and Gly increased significantly. Overall, these findings

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suggested that the excess of soluble Thr could be catabolized into Ser in transgenic seeds via

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3.3 Thr metabolism

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the activity of Thr aldolase, one of the Thr catabolic enzymes [55]. Finally, the authors detected

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higher levels of major nitrogen-containing amino acids such as Glu, Asn, Arg and Gln. Despite

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these dramatic metabolic rearrangements, the transgenic soybean seeds appeared normal and

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germinated well under greenhouse conditions [55].

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BCAAs and their respective products contribute to the growth, defense and production of food

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flavor components in plants (reviewed in [9]). Their catabolism was also shown to provide an

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3.4 Val, Ile and Leu metabolism

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demonstrated that BCAAs accumulate in the seeds of Arabidopsis mutants having defects in

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each of two enzymes in the biosynthetic pathway of these amino acids [16]. These genes are

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Thr deaminase (omr1 mutant) and acetohydroxyacid synthase small subunit 2 (ahass2 mutant).

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Consistent with an in vivo role of Thr deaminase in seed Ile homeostasis, the mutant seeds had

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up to a 8.9-fold increase in Ile compared to the WT, and the levels of Val and Leu increased

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up to 3.7-fold. Higher levels of total Val and Leu, up to 5.5- fold, were also found in the ahass2-

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alternative source of energy under long-term dark conditions [56]. Previous studies

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1D mutant [16]. These data provide evidence of a similar regulatory hierarchy of these

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committed enzymes in seed BCAA homeostasis, which is related to the fact that all BCAAs

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share four biosynthetic enzymes [57].

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in the degradation steps of BCAAs. Notably, the higher BCAA levels were associated in seeds

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with an elevation of additional amino acids [58-60]. For example, an isovaleryl-coenzyme A

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dehydrogenase null mutant showed elevated levels of BCAAs and nine other FAAs in seeds,

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but not in leaves [59, 61]. Similarly, mutants having defects in hydroxylmethylglutaryl-CoA

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lyase and 3-methylcrotonyl-CoA accumulated higher levels of BCAA and 10 other FAAs in

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seeds [62], and many other global effects on seed development and germination. These

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Higher BCAA levels were also found in mutant seeds of Arabidopsis lines that are blocked

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examples highlighted the complex and vital roles of the metabolism of BCAAs in seeds versus

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other tissues. The degradation and breakdown products of BCAAs serve as an alternative

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source of energy since they could be re-directed and enter the TCA cycle and/or donate

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BCAAs serve as respiratory substrates in various processes [56]. The last biosynthetic enzyme

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electrons directly to the electron transfer flavoprotein machinery [57, 61]. Additionally,

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seeds with the natural variation of BCAAs, as found by examining 313 Arabidopsis accessions

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of BCAAs, the branch-chain amino acid transferase2 (BCAT2), was found to be associated in

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addition, this study showed positive correlations between the expression levels of genes

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involved in the BCAA catabolism to those participating in stress responses, developmental

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processes and the circadian clock mechanism [58].

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[58]. This gene uniquely shows strongly induced expression in late seed maturation. In

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The pathway leading to the biosynthesis of His had a great impact on seed development and

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behavior. Previous studies showed that mutations in His biosynthesis number 1A enzyme,

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mainly expressed in embryo tissues, led to altered regulation and accumulation of storage

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compounds in seeds [63, 64]. These effects were mostly attributed to the altered expression of

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genes participating in β-oxidation and ABA biosynthesis in the seeds of the hisn1a mutant.

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Both of these processes have great impacts on seed maturation and germination: the β-oxidation

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pathway is a catabolic process where fatty acids are broken down to generate acetyl-CoA and

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co-enzymes fueling the TCA cycle and electron transport chain, while the ABA signal

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transduction cascade activates genes required for the synthesis of seed-storage reserves and the

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3.5 His metabolism

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acquisition of desiccation tolerance [65]. Accordingly, hisn1a mutant seeds accumulated

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significantly lower oil contents but much higher levels of seed-storage proteins [66].

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Alternatively, the mutation of ABA-deficient 2 (ABA2) blocked these effects of His on β-

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assays showed that a putative His-binding domain was present in the general control non-

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derepressible2 (GCN2) protein, an important amino acid sensor in plants, suggesting that His

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may serve as a signal molecule in seeds capable of regulating plant metabolic homeostasis [66].

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Despite these lines of evidence, the effects of His on the levels of other amino acids remain

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oxidation processes, inferring that ABA mediates this mode of regulation. Further structural

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of genes involved in the biosynthesis of the amino acids His, Lys, Try and Phe [67]. The soluble

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pool of Ala, Asp, Glu, Pro, Thr, Phe, Try, Trp and Val increased between 1.5- and 2-fold

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unclear. For example, His starvation in Arabidopsis plants was found to increase the expression

compared to seeds of non-treated plants [67]. Conversely, an excess of His resulting from

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constitutive expression of the HISN1B gene in Arabidopsis had little or no effect on the

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concentrations of any other amino acids [64]. A correlation between His and the concentrations

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of eight other minor amino acids has been shown in potato shoot (Solanum tuberosum), while

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by the cat4 mutant lines that harbor a mutation in His transporter, which transfers His from the

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vacuole to the cytosol, exhibited over-accumulation of His but no other amino acids [68]. In

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light of these lines of evidence, future studies are required to better understand the metabolic

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regulatory roles of His in developing seeds, and particularly its impacts on the metabolism of

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other amino acids.

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in wheat (Triticum aestivum), no such correlation was found [64]. Moreover, seeds produced

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3.6 Pro metabolism

352 353 354

Arabidopsis, for example, Pro represents up to 26% of the total FAA pool, while in vegetative

355

U

Pro, a non-essential amino acid, accumulates in large amounts in plant seeds [69]. In

N

tissues, it accounts for only 1-3% [70]. It seems that such large contents in seeds cannot only

A

be attributed to an increased demand of storage protein synthesis. One possible explanation

M

was the important roles played by Pro in conferring desiccation tolerance to seeds since Pro

356 357 358 359

stress conditions. Furthermore, the degradation products of Pro are essential for generating

360

energy for metabolically-demanding cells [71]. Pro also plays additional roles in embryo

361

development [70]. One of the rate-limiting enzymes involved in Pro biosynthesis, pyrroline 5-

362

carboxylate synthetase (P5CS), was shown to be highly transcribed throughout embryogenesis

363

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PT

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serves both as an osmoprotectant and a redox-buffering agent with antioxidant capacities under

364

defective embryos. Attempts to rescue these mutant embryos via the supply of Pro failed,

365

therefore the authors concluded that the availability of endogenous Pro during early

366

A

in Arabidopsis seeds [72]. Consistently, a mutant harboring a mutation in P5CS produced

embryogenesis through P5CS activity is critical for normal seed development [73]. In addition,

367

lowered expression levels of AtP5CS1 caused inferior Pro concentration during seed

368

germination and delayed the emergence of the radicle compared to WT seeds, which had an

369

ongoing synthesis of Pro and normal germination rates [74].

370

16

371

The level of GABA accumulates during the maturation stage of seed development, and is

372

therefore suggested to play a role in seeds [2, 5]. GABA is mainly formed by the degradation

373

of Glu by the activity of glutamate decarboxylase (GAD) enzyme. Previous studies

374

demonstrated a rapid formation of GABA in developing seeds, which then degrades to

375

succinate on the onset of germination [75]. Thus, it was suggested that the accumulation of

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376

GABA in the mature dry seed supplies available energy for germination [76]. To elucidate the

377

metabolic regulatory roles of GAD in seed development, a truncated Petunia hybrida GAD,

378

missing the carboxyl-terminal regulatory calmodulin-binding domain, was expressed in

379

Arabidopsis under the control of the seed-specific phaseolin promoter [76]. Mature seeds

380

U

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3.7 GABA metabolism

N

produced by these transgenic lines accumulated considerable amounts of GABA, as well as

A

higher levels of Asp, Asn, Met, Cys, Ser and Trp [76]. Similarly, higher levels of other amino

M

acids were also produced in GABA-enriched rice grains expressing a truncated version of the

381 382 383 384

displayed up to 47% higher protein content compared to WT [76]. The increase in overall

385

content of FAAs was accompanied by a depletion of TCA cycle intermediates and a decrease

386

in total fatty acids content [76]. Later, the authors utilized labeling experiments to show that

387

GABA strongly associates with transcriptional and metabolic processes occurring in early seed

388

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OsGAD2 [77]. Corresponding to its general effects on amino acids, transgenic mature seeds

389

metabolic roles, it was recently suggested that GABA serves as a signaling molecule for altered

390

cycling of TCA intermediates by eliciting changes in the electric potential across membranes.

391

A

germination and contributes to the biosynthesis of amino acids in seeds [76]. Apart from its

These effects are mediated by GABA-regulated aluminum-activated malate transporter [78]

392

that was suggested to act a receptor for GABA [79]. Taken together, the biosynthesis of GABA

393

chiefly through the activity of GAD, plays an important role in regulating carbon-to-nitrogen

394

balance and storage reserve accumulation during seed development and germination by

395

17

modulating the flux of carbon and energy through the TCA cycle [22]. It also considered as a

396

legitimate signaling molecule in plants controlling different process in plants [27, 79].

397 398

4. Altered levels of certain amino acids can greatly impact the metabolism

of other amino acids in seeds

399

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400

401

Arabidopsis seeds led to dramatic changes in the levels of other amino acids (summarized in

402

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The studies described above have shown that altered levels of specific amino acids in

403

amino acids [59], those of Met with 19 [12], GABA with six [76], and His with 12 amino acids

404

[64]. Hence, these observations strongly infer that the metabolic networks of the amino acid

405

biosynthesis are much more interconnected at least in Arabidopsis seeds than previously

406

assumed [40, 68]. These phenomena, however, are apparently broader and were also observed

407

in tomato seeds. Metabolite profiling and network analysis that analyzed a collection of 76

408

tomato introgression lines implicated six highly co-regulated amino acids, Gly, Ser, Thr, Ile,

409

ED

M

A

N

U

Table 1). For example, higher levels of Leu were associated with elevated quantities of 11 other

410

suggested to form a tightly inter-regulated module, acting as the backbone of this metabolic

411

PT

Val and Pro, with an average r-value of 0.87 [10]. Moreover, these six amino acids were

412

tissues [10]. The significant positive correlations between amino acid levels reflect a highly

413

regulated amino acid metabolism that is apparently mediated by their incorporation rates into

414

the seed-storage proteins. The results also suggested that seed amino acids play a central hub-

415

A

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network that was specific to the seeds and was not detected in the fruit pericarp and leave

like role in the core of its metabolic network, thus maintaining high interactions with other

416

metabolite groups such as organic acids and sugars [10].

417

In the previous section we described various studies that reported of elevated levels of some

418

FAAs in seeds when genetic manipulations were performed in the biosynthetic pathway of

419

some specific amino acid (Table 1). FAA elevations during the development of these transgenic

420

18

421

rule out that metabolic rearrangements in other metabolic groups contributed to the elevated

422

FAA pool in the transgenic seeds and that FAA elevations randomly occurred. Still, the studies

423

in Arabidopsis and tomato seeds provide a strong foundation that FAAs form a highly

424

interconnected metabolic cluster. Thus, these propose the possibility of global factors that

425

regulate the biosynthesis of FAAs and/or accumulations as a whole in plant seeds. Evidences

426

for such a tight regulation of FAA pool were partially described in plants so far. For instance,

427

Arabidopsis leaves grown under abiotic stresses exhibited significantly induced expression of

428

FAA catabolic genes but only minor changes in the expression level of genes operate in their

429

biosynthetic pathways [80]. This phenomena was named "gene coordination" that was later

430

U

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seeds might also be affected by the differential levels of other metabolites. Thus, we cannot

N

also shown to exist in FAAs belong to the Asp-family in Arabidopsis such as Lys, Met, Thr,

A

Ile and Gly, as well as in the group of aromatic amino acids comprising Trp, Phe and Tyr [81].

M

The syntheses of these families of FAAs start from one major precursor, but their biosynthetic

431 432 433 434

regulatory mechanisms. Under optimal growth conditions all branches are expected to operate

435

ED

pathways then divide into different branches that are likely coordinated by constricted

436

However, under specific stress conditions, metabolic fluxes of certain FAAs are expected to

437

PT

efficiently to allow the synthesis of FAAs for their incorporation into storage-proteins.

438

adjustments [81]. One example is the synchronized expression of 7 genes controlling Met

439

levels and its derivatives in the Asp-family pathway [81]. Such evidences are evidently rare

440

and thus urging for future work in order to define yet unidentified global regulators of FAA

441

A

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increase in some branches at the expense of other branches to allow optimal metabolic

442

metabolism in seeds. Some evidences for such common regulation of FAAs were already isolated and

443

characterized in yeast and in mammals. Amino acid starvation in yeasts was shown to initiates

444

a signal transduction cascade starting with the activation of GCN4 transcription factor, which

445

19

446

signal transduction networks were shown to tightly regulate the supply of FAAs, the

447

mammalian target of rapamycin complex 1 (mTORC1) pathway and the integrated stress

448

response (ISR). The activities of these two cascades with respect to FAA biosynthesis are

449

highly associated to maintain a spectrum of cellular responses ranging from the induction of

450

growth to activation of cell death [83]. It is therefore highly logical that similar ‘master’

451

transcription factors and/or regulatory networks also operate in plants and especially in seeds

452

to coordinate the accumulation of FAAs. However, no such regulators were reported in plants

453

to date. Undoubtedly, revealing these mechanisms will enable the scientific community with

454

more founded and sophisticated tools to increase the levels of FAAs in seeds, and consequently

455

U

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in turn activates the transcription of FAA biosynthetic genes [82]. In mammals two major

A

N

their protein contents and nutritional properties.

M

5. Conclusions and future prospective

ED

Recent studies investigating a large collection of mutants and/or transgenic plants with

456 457 458

459 460

seeds showed that FAAs play central ‘hub-like’ roles in the seed’s metabolic network. The

461

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different genetic manipulations in key enzymes involved in the metabolism of amino acids in

462

biosynthetic pathway of various biochemical groups, such as sugars, organic acids and many

463

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accumulated knowledge infers that FAAs tend to maintain strong interactions with the

464

excellent source of energy, the pool of FAAs was significantly involved in seed maturation and

465

early germination processes. As a result, when the levels of one or more amino acids are

466

changed due to natural responses, environmental cues or artificial genetic manipulations, they

467

have immense effects on seed vigor and physiology, as well as on nutritional values.

468

A

others [2, 10, 68]. By contributing to the synthesis of seed-storage proteins and serving as an

Despite the considerable data accumulated in recent years regarding the functional

469

regulatory roles of FAAs in seed development, their fine accumulation patterns and their

470

21

471

transcriptomics, proteomics, metabolomics as well as feeding and flux experiments. The

472

combination of such ’omics’ strategies will assist in revealing the complex metabolic networks

473

involved in FAA and TAA metabolism and will shed light on the factors that regulate their

474

levels in seeds. Several key questions remain unanswered with respect to these metabolic

475

networks: (i) Are there ‘master genes’ and/or regulators that control the levels of FAA in seeds?

476

(ii) What is the nature of association between the biosynthetic pathways leading to the

477

formation of FAAs during seed development? (iii) Which mechanisms operate to transport

478

FAAs from vegetative towards reproductive tissues on the onset of seed development? and (iv)

479

How do changes in the levels of FAAs affect the metabolism of unrelated biochemical groups

480

U

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IP T

metabolism during seed development require further investigations. These should include

N

and storage reserves? Once the scientific community will overcome these unanswered

A

questions, it will be possible to perform more rational genetic manipulations in the biosynthetic

482 483 484 485 486

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Acknowledgments

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and increase seed fitness on the other.

M

pathways of FAAs in seeds that could potentially increase its nutritional values on the one hand

481

487

Foundation (grant no. 1004/15).

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The research on the metabolism of amino acids in seed is supported by the Israel Science

A

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21

References

490

[1] S. Baud, J.P. Boutin, M. Miquel, L. Lepiniec, C. Rochat, An integrated overview of seed

491

development in Arabidopsis thaliana ecotype WS, Plant Physiol. Biochem. 40 (2002) 151-

492

160.

493

IP T

]2[ A. Fait, R. Angelovici, H. Less, I. Ohad, E. Urbanczyk-Wochniak, A.R. Fernie, G. Galili,

494 495

metabolic switches, Plant Physiol. 142 (2006) 839-854.

496

SC R

Arabidopsis seed development and germination is associated with temporally distinct

497

Profiles and Tissue-Specific Source Effects on the Metabolome of Developing Seeds of

498

Brassica napus, PloS one, 10 (2015) e0124794.

499

U

]3[ H. Tan, Q. Xie, X. Xiang, J. Li, S. Zheng, X. Xu, H. Guo, W. Ye, Dynamic Metabolic

A

Annu. Rev. Plant Biol. 56 (2005) 253-272.

N

]4[ H. Weber, L. Borisjuk, U. Wobus, Molecular physiology of legume seed development,

M

]5[ R. Angelovici, G. Galili, A.R. Fernie, A. Fait, Seed desiccation: a bridge between maturation and germination, Trends Plant Sci. 15 (2010) 211-218.

500 501 502 503 504

Westgate, Z. Arendsee, V. Iyer, J. Shanks, B. Nikolau, E.S. Wurtele, A systems biology

505

PT

ED

]6[ L. Li, M. Hur, J.Y. Lee, W. Zhou, Z. Song, N. Ransom, C.Y. Demirkale, D. Nettleton ,M.

506

3 (2015) S9.

507

CC E

approach toward understanding seed composition in soybean, BMC Genomics, 16 Suppl

508

Deciphering gene regulatory networks that control seed development and maturation in

509

Arabidopsis, Plant J. 54 (2008) 608-620.

510

A

]7[ M. Santos-Mendoza, B. Dubreucq, S. Baud, F. Parcy, M. Caboche, L .Lepiniec,

[8] G. Galili, R. Amir, Fortifying plants with the essential amino acids lysine and methionine to improve nutritional quality, Plant Biotechnol. J. 11 (2013) 211-222. [9] G. Galili, R. Amir, A.R. Fernie, The Regulation of Essential Amino Acid Synthesis and Accumulation in Plants, Annu. Rev. Plant Biol. 67 (2016)853-871 22

511 512 513 514

[10] D. Toubiana, Y. Semel, T. Tohge, R. Beleggia, L. Cattivelli, L. Rosental, Z. Nikoloski, D.

515

Zamir, A.R. Fernie, A. Fait, Metabolic profiling of a mapping population exposes new

516

insights in the regulation of seed metabolism and seed, fruit ,and plant relations, PLoS

517

Genetics. 8 (2012) e1002612.

518 519

acid analysis of two maize threonine-overproducing, lysine-insensitive aspartate kinase

520

mutants, Theor. Appl. Genet. 89 (1994) 767-774.

521

IP T

[11] G.J. Muehlbauer, B.G. Gengenbach, D.A. Somers, C.M. Donovan, Genetic and amino-

522

insensitive form of CYSTATHIONINE-gamma-SYNTHASE in Arabidopsis stimulates

523

metabolic and transcriptomic responses associated with desiccation stress, Plant Physiol.

524

U

SC R

[12] H. Cohen, H. Israeli, I. Matityahu, R. Amir, Seed-specific expression of a feedback-

N

166 (2014) 1575-1592.

A

[13] A. Frank, H. Cohen, D. Hoffman, R. Amir, Methionine and S-methylmethionine exhibit

acids, 47 (2015) 497-510.

M

temporal and spatial accumulation patterns during the Arabidopsis life cycle, Amino

ED

[14] L. Gutierrez, O. Van Wuytswinkel, M. Castelain, C. Bellini, Combined networks regulating seed maturation, Trends Plant Sci, 12 (2007) 294-300.

PT

[15] R. Angelovici, A. Fait, A.R. Fernie, G. Galili, A seed high-lysine trait is negatively

525 526 527 528 529 530 531 532

Phytol. 189 (2011) 148-159.

533

CC E

associated with the TCA cycle and slows down Arabidopsis seed germination, New

[16] A. Xing, R.L. Last, A Regulatory Hierarchy of the Arabidopsis Branched-Chain Amino

A

Acid Metabolic Network, Plant Cell, 29 (2017) 1480-1499.

534 535

[17] S. Baud, B. Dubreucq, M. Miquel, C. Rochat, L. Lepiniec, Storage reserve accumulation

536

in Arabidopsis: metabolic and developmental control of seed filling, The Arabidopsis

537

book, 6 (2008) e0113.

538

23

]81[M.J. Hills, Control of storage-product synthesis in seeds, Curr. Opin. Plant Biol. 7 (2004)

539 540

302-308.

541

amino acid profiles suggest a possible role for asparagine in the control of storage-product

542

accumulation in developing seeds of low- and high-protein soybean lines, J. Exp. Bot. 56

543

(2005) 1951-1963.

544

IP T

]82[C. Hernandez-Sebastia, F. Marsolais, C. Saravitz, D. Israel, R.E. Dewey, S.C. Huber, Free

545

Oecking, Desulfoglucosinolate sulfotransferases from Arabidopsis thaliana catalyze the

546

final step in the biosynthesis of the glucosinolate core structure, J. Biol. Chem. 279 (2004)

547

50717-50725.

548

SC R

]22[M. Piotrowski, A. Schemenewitz, A. Lopukhina, A. Muller, T. Janowitz, E.W. Weiler, C.

N

U

]28[S. Slavov, H. van Onckelen, R. Batchvarova, A. Atanassov, E. Prinsen, IAA production during germination of Orobanche spp. seeds, J. Plant Physiol.161 (2004) 847-853.

M

A

]22[A. Fait, H. Fromm, D. Walter, G. Galili, A.R. Fernie, Highway or byway: the metabolic role of the GABA shunt in plants, Trends Plant Sci, 13 (2008) 14-19.

ED

]23[S. Dam, B.S. Laursen, J.H. Ornfelt, B. Jochimsen, H.H. Staerfeldt, C. Friis, K. Nielsen,

549 550 551 552 553 554

Stougaard, The proteome of seed development in the model legume Lotus japonicus, Plant

555

PT

N. Goffard, S. Besenbacher, L. Krusell, S. Sato, S. Tabata, I.B. Thogersen, J.J. Enghild, J.

556

Physiol. 149 (2009) 1325-1340.

557

CYSTATHIONINE gamma-SYNTHASE in Seeds Recruits the S-Methylmethionine

558

Cycle, Plant Physiol. 174 (2017) 1322-1333.

559

A

CC E

]24[H. Cohen, Y. Hacham, I. Panizel, I. Rogachev, A. Aharoni, R. Amir, Repression of

]25[M. Watanabe, S. Balazadeh, T. Tohge, A. Erban, P. Giavalisco, J. Kopka, B. Mueller-

560

Roeber, A.R. Fernie, R. Hoefgen, Comprehensive dissection of spatiotemporal metabolic

561

shifts in primary, secondary, and lipid metabolism during developmental senescence in

562

Arabidopsis, Plant Physiol.162 (2013) 1290-1310.

563

24

[26] M. Tegeder, C. Masclaux-Daubresse, Source and sink mechanisms of nitrogen transport

564 565

and use, New Phytol, 217 (2018) 35-53. [27] M. Tegeder, U.Z. Hammes, The way out and in: phloem loading and unloading of

566 567

amino acids, Current Opin Plant Biol, 43 (2018) 16-21. [28] C. Masclaux-Daubresse, F. Daniel-Vedele, J. Dechorgnat, F. Chardon, L. Gaufichon, A.

568 569

sustainable and productive agriculture, Ann Bot, 105 (2010) 1141-1157.

570

IP T

Suzuki, Nitrogen uptake, assimilation and remobilization in plants: challenges for

571

UMAMIT14 is an amino acid exporter involved in phloem unloading in Arabidopsis roots,

572

J Exp Bot, 67 (2016) 6385-6397.

573

U

SC R

[29] J. Besnard, R. Pratelli, C. Zhao, U. Sonawala, E. Collakova, G. Pilot, S. Okumoto,

N

[30] T. Mae, Nitrogen utilization, growth and yield in rice plants, in: T.Ohyamaand,

A

K.Sueyoshi (Eds.) Nitrogen Assimilation in Plants, Research Signpost, Kerala, 2010, pp.

M

243–253.

574 575 576 577

S.J. Miller, J.M. Baker, P.J. Verrier, J.L. Ward, M.H. Beale, P.B. Barraclough, M.J.

578

ED

[31] J.R. Howarth, S. Parmar, J. Jones, C.E. Shepherd, D.I. Corol, A.M. Galster, N.D. Hawkins,

579

deficiency during wheat grain filling, J. Ex. Bot, 59 (2008) 3675-3689.

580

PT

Hawkesford, Co-ordinated expression of amino acid metabolism in response to N and S

[32] H. Hayashi, M. Chino, Chemical composition of phloem sap from the upper most

CC E

internode of the rice plant., Plant Cell Physiol. 31 (1990) 247–251.

581 582 583

Wang, J. Stougaard, J.M. Vega, A.J. Marquez, The K+-dependent asparaginase, NSE1, is

584

A

[33] A. Credali, M. Garcia-Calderon, S. Dam, J. Perry, A. Diaz-Quintana, M. Parniske, T.L.

crucial for plant growth and seed production in Lotus japonicus, Plant Cell Physiol. 54

585

(2013) 107-118.

586

25

[34] L. Zhang, M.G. Garneau, R. Majumdar, J. Grant, M. Tegeder, Improvement of pea

587

biomass and seed productivity by simultaneous increase of phloem and embryo loading

588

with amino acids, Plant J, 81 (2015) 134-146.

589

[35] J. Schwender, Y. Shachar-Hill, J.B. Ohlrogge, Mitochondrial metabolism in developing

591

embryos of Brassica napus, J Biol Chem, 281 (2006) 34040-34047.

IP T

[36] R. Pratelli, G. Pilot, Regulation of amino acid metabolic enzymes and transporters in plants, J Exp Bot, 65 (2014) 5535-5556.

590

592 593 594

Grotemeyer, D. Rentsch, E. Truernit, F. Ladwig, A. Bleckmann, T. Dresselhaus, U.Z.

595

Hammes, Amino Acid Export in Developing Arabidopsis Seeds Depends on UmamiT

596

N

Facilitators, Current Biol. 25 (2015) 3126-3131.

U

SC R

[37] B. Muller, A. Fastner, J. Karmann, V. Mansch, T. Hoffmann, W. Schwab, M. Suter-

A

[38] W. Wang, M. Xu, G. Wang, G. Galili, New insights into the metabolism of aspartate-

M

family amino acids in plant seeds, Plant Reprod on, (2018).

597 598 599 600

synergistically boosts lysine content and also transregulates the metabolism of other amino

601

ED

[39] X. Zhu, G. Galili, Increased lysine synthesis coupled with a knockout of its catabolism

acids in Arabidopsis seeds, Plant Cell, 15 (2003) 845-853.

PT

[40] R. Angelovici, A. Fait, X. Zhu, J. Szymanski, E. Feldmesser, A.R. Fernie, G. Galili,

602 603 604

during Arabidopsis seed development, Plant Physiol, 151 (2009) 2058-2072.

605

CC E

Deciphering transcriptional and metabolic networks associated with lysine metabolism

606

catabolism in both reproductive and vegetative tissues, Plant Physiol, 135 (2004) 129-136.

607

A

[41] X. Zhu, G. Galili, Lysine metabolism is concurrently regulated by synthesis and

[42] Y. Hacham, G. Schuster, R. Amir, An in vivo internal deletion in the N-terminus region

608

of Arabidopsis cystathionine gamma-synthase results in CGS expression that is insensitive

609

to methionine, Plant J, 45 (2006) 955-967.

610

26

[43] H. Cohen, O.M. Shir, Y. Yu, W. Hou, S. Sun, T. Han, R. Amir, Genetic background and

611

environmental conditions drive metabolic variation in wild type and transgenic soybean

612

(Glycine max) seeds, Plant, Cell Environ. 39 (2016) 1805-1817.

613 614

Ishimoto, Differential response of methionine metabolism in two grain legumes, soybean

615

and azuki bean, expressing a mutated form of Arabidopsis cystathionine gamma-synthase,

616

J Plant Physiol, 170 (2013) 338-345.

617

IP T

[44] M.S. Hanafy, S.M. Rahman, Y. Nakamoto, T. Fujiwara, S. Naito, K. Wakasa, M.

618

Amir, Soybean seeds expressing feedback-insensitive cystathionine γ-synthase exhibit a

619

higher content of methionine., J Exp Bot, 64 (2013) 1917-1926.

620

U

SC R

[45] S. Song, W. Hou, I. Godo, C. Wu, Y. Yu, I. Matityahu, Y. Hacham, S. Sun, T. Han, R.

N

[46] I. Matityahu, I. Godo, Y. Hacham, R. Amir, Tobacco seeds expressing feedback-

A

insensitive cystathionine gamma-synthase exhibit elevated content of methionine and

M

altered primary metabolic profile, BMC Plant Biol, 13 (2013) 206.

621 622 623 624

transcriptome and metabolome of transgenic Arabidopsis seeds, Plant Cell Rep, 36 (2017)

625

ED

[47] H. Cohen, R. Amir, Dose-dependent effects of higher methionine levels on the

719-730.

626

PT

[48] H. Cohen, A. Pajak, S. Pandurangan, R. Amir, F. Marsolais, Higher endogenous

627 628

and lipids, Amino acids, 48 (2016) 1413-1422.

629

CC E

methionine in transgenic Arabidopsis seeds affects the composition of storage proteins

630

Arabidopsis thaliana HMT2, a methionine biosynthetic enzyme, increases seed

631

A

[49] M. Lee, T. Huang, T. Toro-Ramos, M. Fraga, R.L. Last, G. Jander, Reduced activity of

methionine content, Plant J, 54 (2008) 310-320.

632

[50] H.C. Nguyen, R. Hoefgen, H. Hesse, Improving the nutritive value of rice seeds: elevation

633

of cysteine and methionine contents in rice plants by ectopic expression of a bacterial

634

serine acetyltransferase, J Exp Bot, 63 (2012) 5991-6001.

635

27

[51] J. Planta, X. Xiang, T. Leustek, J. Messing, Engineering sulfur storage in maize seed

636 637

proteins without apparent yield loss, PANS, 114 (2017) 11386-11391. [52] Y. Hacham, I. Matityahu, G. Schuster, R. Amir, Overexpression of mutated forms of

638

aspartate kinase and cystathionine gamma-synthase in tobacco leaves resulted in the high

639

accumulation of methionine and threonine, Plant J, 54 (2008) 260-271.

640 641

Additive effects of the feed-back insensitive bacterial aspartate kinase and the Brazil nut

642

2S albumin on the methionine content of transgneic narbon bean (Vicia narbonensis L.),

643

Mol Breed, 11 (2003) 187-201.

644

SC R

IP T

[53] D. Demidov, C. Horstmann, M. Meixner, T. Pickard, I. Saalbach, G. Galili, K. Muntz,

U

[54] H. Karchi, O. Shaul, G. Galili, Seed specific expression of a bacterial desensitized

N

aspartate kinase increases the production of seed threonine and methionine in transgenic

A

tobacco., Plant J. 3 (1993) 721-727.

M

[55] Q. Qi, J. Huang, J. Crowley, L. Ruschke, B.S. Goldman, L. Wen, W.D. Rapp,

645 646 647 648 649

characterization and seed-specific expression of lysine-insensitive variants of aspartate

650

ED

Metabolically engineered soybean seed with enhanced threonine levels: biochemical

651

193-204.

652

PT

kinases from the enteric bacterium Xenorhabdus bovienii, Plant Biotechnol J, 9 (2011)

653

Dehydrogenase Complex on Amino Acid Homeostasis in Arabidopsis, Plant Physiol, 169

654

(2015) 1807-1820.

655

CC E

[56] C. Peng, S. Uygun, S.H. Shiu, R.L. Last, The Impact of the Branched-Chain Ketoacid

A

[57] S. Binder, Branched-Chain Amino Acid Metabolism in Arabidopsis thaliana, The

656 657

Arabidopsis book, 8 (2010) e0137.

[58] R. Angelovici, A.E. Lipka, N. Deason, S. Gonzalez-Jorge, H. Lin, J. Cepela, R. Buell,

658

M.A. Gore, D. Dellapenna, Genome-wide analysis of branched-chain amino acid levels in

659

Arabidopsis seeds, Plant Cell, 25 (2013) 4827-4843.

660

28

[59] L. Gu, A.D. Jones, R.L. Last, Broad connections in the Arabidopsis seed metabolic

661

network revealed by metabolite profiling of an amino acid catabolism mutant, Plant J. 61

662

(2010) 579–590.

663

[60] C. Lu, J. Kang, Generation of transgenic plants of a potential oilseed crop Camelina sativa by Agrobacterium-mediated transformation, Plant Cell Rep, 27 (2008) 273-278.

664 665 666

Obata, N. Schauer, I.A. Graham, C.J. Leaver, A.R. Fernie, Identification of the 2-

667

hydroxyglutarate and isovaleryl-CoA dehydrogenases as alternative electron donors

668

linking lysine catabolism to the electron transport chain of Arabidopsis mitochondria,

669

Plant Cell, 22 (2010) 1549-1563.

670

U

SC R

IP T

[61] W.L. Araujo, K. Ishizaki, A. Nunes-Nesi, T.R. Larson, T. Tohge, I. Krahnert, S. Witt, T.

N

[62] G. Ding, P. Che, H. Ilarslan, E.S. Wurtele, B.J. Nikolau, gucarboxylase indicates a

A

complex role for mitochondrial leucine catabolism during seed development and

M

germination, Plant J. 70 (2012) 562-577.

[63] R. Muralla, C. Sweeney, A. Stepansky, T. Leustek, D. Meinke, Genetic dissection of

ED

histidine biosynthesis in Arabidopsis, Plant Physiol, 144 (2007) 890-903. [64] A. Stepansky, T. Leustek, Histidine biosynthesis in plants, Amino acids, 30 (2006) 127-

672 673 674 675 676 677

PT

142.

671

[65] O. Leprince, A. Pellizzaro, S. Berriri, J. Buitink, Late seed maturation: drying without

678 679

CC E

dying, J. Exp. Bot, 68 (2017) 827-841.

[66] H. Ma, S. Wang, Histidine Regulates Seed Oil Deposition through Abscisic Acid

A

Biosynthesis and beta-Oxidation, Plant Physiol. 172 (2016) 848-857.

[67] D. Guyer, D. Patton, E. Ward, Evidence for cross-pathway regulation of metabolic gene expression in plants, Proc Natl Acad Sci U S A, 92 (1995) 4997-5000.

29

680 681 682 683

[68] R. Angelovici, A. Batushansky, N. Deason, S. Gonzalez-Jorge, M.A. Gore, A. Fait, D.

684

DellaPenna, Network-Guided GWAS Improves Identification of Genes Affecting Free

685

Amino Acids, Plant Physiol, 173 (2017) 872-886.

686

[69] R. Mattioli, P. Costantino, M. Trovato, Proline accumulation in plants: not only stress,

687 688

Plant signaling & behavior, 4 (2009) 1016-1018.

Arabidopsis reproductive development, BMC Plant Biol, 12 (2012) 191.

IP T

[70] D. Funck, G. Winter, L. Baumgarten, G. Forlani, Requirement of proline synthesis during

689 690 691

tolerance or is proline homeostasis a more critical issue?, Plant Cell Environ. 37 (2014)

692

300–311.

693

U

SC R

[71] P.B.K. Kishor, N. Sreenivasulu, Is proline accumulation per se correlated with stress

N

[72] G. Szekely, E. Abraham, A. Cseplo, G. Rigo, L. Zsigmond, J. Csiszar, F. Ayaydin, N.

A

Strizhov, J. Jasik, E. Schmelzer, C. Koncz, L. Szabados, Duplicated P5CS genes of

M

Arabidopsis play distinct roles in stress regulation and developmental control of proline biosynthesis, Plant J, 53 (2008) 11-28.

ED

[73] D. Meinke, R. Muralla, C. Sweeney, A. Dickerman, Identifying essential genes in Arabidopsis thaliana, Trends Plant Sci. 13 (2008) 483-491.

PT

[74] P.D. Hare, W.A. Cress, J. van Staden, A regulatory role for proline metabolism in

694 695 696 697 698 699 700 701

[75] L.G. Tuin, A.W. Shelp, In situ [14C]Glu metabolism by developing soybean cotyledons.

702

CC E

stimulating Arabidopsis thaliana seed germination, Plant Growth Regul. 39 (2003) 41-50.

I. Metabolic routes, J. Plant Physiol, 143 (1994) 1-7.

A

[76] A. Fait, A.N. Nesi, R. Angelovici, M. Lehmann, P.A. Pham, L. Song, R.P. Haslam, J.A.

703 704

Napier, G. Galili, A.R. Fernie, Targeted enhancement of glutamate-to-gamma-

705

aminobutyrate conversion in Arabidopsis seeds affects carbon-nitrogen balance and

706

storage reserves in a development-dependent manner, Plant Physiol, 157 (2011) 1026-

707

1042.

708

31

[77] K. Akama, J. Kanetou, S. Shimosaki, K. Kawakami, S. Tsuchikura, F. Takaiwa, Seed-

709

specific expression of truncated OsGAD2 produces GABA-enriched rice grains that

710

influence a decrease in blood pressure in spontaneously hypertensive rats, Transgenic Res,

711

18 (2009) 865-876.

712 713

S. Wege, S. Shabala, J.A. Feijo, P.R. Ryan, M. Gilliham, GABA signalling modulates

714

plant growth by directly regulating the activity of plant-specific anion transporters,

715

Nature Commun, 6 (2015) 7879.

716

SC R

IP T

[78] S.A. Ramesh, S.D. Tyerman, B. Xu, J. Bose, S. Kaur, V. Conn, P. Domingos, S. Ullah,

[79] M. Gilliham, S.D. Tyerman, Linking metabolism to membrane signaling: The GABA-

U

malate connection, Trends Plant Sci, 21 (2016) 295-301.

N

[80] H. Less, G. Galili, Principal transcriptional programs regulating plant amino acid

718 719 720 721

genome-wide system interactions of the Asp-family and aromatic amino acid networks of

722

amino acid metabolism in plants, Amino Acids, 39 (2010) 1023-1028.

723

ED

[81] H. Less, R. Angelovici, V. Tzin, G. Galili, Principal transcriptional regulation and

M

A

metabolism in response to abiotic stresses, Plant Physiol, 147 (2008) 316-330.

717

[82] A. Sundaram, C.M. Grant, A single inhibitory upstream open reading frame (uORF) is

PT

sufficient to regulate Candida albicans GCN4 translation in response to amino acid starvation conditions, RNA, 20 (2014) 559-567.

CC E

[83] T.G. Anthony, Homeostatic responses to amino acid insufficiency, Animal Nut, 1 (2015)

725 726 727 728

135-137.

[84] H. Karchi, D. Miron, S. Ben-Yaacov, G. Galili, The lysine-dependent stimulation of

A

724

729

lysine catabolism in tobacco seed requires calcium and protein phosphorylation, Plant

730

Cell, 7 (1995) 1963-1970.

731

31

[85] X. Xiang, Y. Wu, J. Planta, J. Messing, T. Leustek, Overexpression of serine

732

acetyltransferase in maize leaves increases seed-specific methionine-rich zeins, Plant

733

Biotechnol J, 16 (2018) 1057-1067.

734

A

CC E

PT

ED

M

A

N

U

SC R

IP T

735

32

736

Figure 1. Schematic representation of amino acid biosynthesis pathways in plants. Only

737

several enzymes and metabolites are specified. Enzymes are indicated in red text. Dashed

738

arrowed lines represent more than one enzymatic step. Abbreviations for metabolites: G6P,

739

glucose 6-phosphate; 3PG, 3-phosphoglyverate; R5P, Ribose 5-phosphate; OPH, O-

740

IP T

Figure captions

741

for enzymes: AK, Asp kinase; CGS, cystathionine γ-synthase; DHDPS, dihydrodipicolinate

742

SC R

phosphohomoserine; SMM, S-methylMet; ETF, electron transfer flavoprotein. Abbreviations

743

dehydrogenase; P5CS, pyrroline 5-carboxylate synthetase; GAD, glutamate decarboxylase;

744

HISN, His biosynthetic ; HASS, acetohydroxyacid synthase small subunit; BCAT, branch-

745

chain amino acid transferase; HMT, homocysteine methyltransferases; SAT, Ser

746

acetyltransferase; APR, 3'-phosphoadenosine-5'-phosphosulfate reductase; LKR/SDH, Lys-

747

ketoglutarate reductase/saccharopine dehydrogenase.

748

CC E

PT

ED

M

A

N

U

synthase; TA, Thr aldolase; TS, Thr synthase; TD, Thr deaminase; IVDH, isovalery-CoA

A

749 750

33

I N U SC R

Table 1. Summary of studies utilizing genetic tools in an effort to increase the levels of several FAAs in seeds and their impact on seed central metabolism, physiology and behavior

752

Amino acid manipulated

Gene manipulated*

Manipulation methodology

FC** of amino acid level vs. WT

Major effects on seeds

Reference

Tobacco

Lys

DHDPS

Seed-specific expression

No significant changes

Increased activity of LKR/SDH enzyme during maturation and desiccation stages of seed development

[84]

Arabidopsis

Lys

DHDPS

Seed-specific expression Seed-specific expression / TDNA insertion mutation

12-fold increase in Lys content

Not determined

[39]

Seed-specific expression

6-fold increase in Met content

CGS

Seed-specific expression

7- and 3.4-fold increase in Met content (ZD and JX lines, respectively)

Over-accumulation of 11 FAAs; Higher and lower contents of total protein and total oil, respectively

[43, 45]

Slight reduction in Met content

Lower levels of Cys and GSH; Total protein content increased by 27%; A slight but significantly reduction in starch content; Insignificant reduction in oil content

[46]

40- fold increase in Met content 1.4- and 4.8-fold increased soluble and

Not determined

[49]

The level of the BCAA increased and of Cys and glutathione

[50]

Arabidopsis

Met

CC E A

Soybean

Met

M

ED

Lys

PT

Arabidopsis

CGS

A

Plant Species

DHDPS / LKR/SDH

Tobacco

Met

CGS

Seed-specific expression

Arabidopsis

Met

HMT2

T-DNA insertion mutation

Rice

Met

SAT

Overexpression

80-fold increase in Lys content

34

751

Reduced levels of Glu and Asp together with increased levels of Gln and Asn; Lower germination rates; Lower level of TCA intermediates; Higher transcript levels of ribosomal proteins Over-accumulation of 19 FAAs (apart from Tyr); Buildup of stress-related metabolites and transcripts; Higher and lower contents of seed-storage proteins and fatty-acids, respectively

[39, 40]

[12, 48]

I Met

SAT

Tobacco

Met, Thr

AK

Soybean

Thr

AK

Arabidopsis

Thr

Arabidopsis

Val, Ile, Leu

PT

CC E

IVD

Leu

MCC

Arabidopsis

His

HISN1A

GABA

GAD

A *

TD

Arabidopsis

Arabidopsis

N U SC R

Maize

Leaf-specific expression Bundle sheath cellspecific promoter

A

APR

Seed-specific expression

M

Met

ED

Maize

Seed-specific expression T-DNA insertion mutation

T-DNA insertion mutation T-DNA insertion mutation T-DNA insertion mutation Seed-specific expression

total Met contents, respectively 57.6% higher Met content 1.4-fold increase in Met content 3-fold increase in Met content; 16-fold increase in Thr content 100-fold increase in Thr content 8.9-fold increase in Ile content; 3.7-fold increases in Val and Leu contents 28-, 30- and 16-fold increases in Leu, Ile and Val contents, respectively 4-fold increase in Leu content 3.2-fold reduction in His content 300- to 1200-fold increases in GABA content

[51]

Higher expression of the Met-rich 10-kDa δ-zein

[85]

Not determined

[54]

Elevation of 9 amino acids and up to 3.5-fold increase in the total FAA content

[55]

Changes in germination rates

[16]

Increased levels of 12 FAAs

[59]

Increased levels of 10 FAAs. Total FAA content increased by 2.5-fold Lower oil contents; Higher seed-storage protein content Transcript phenotype of early seed germination; Over-accumulation of 6 FAAs; Higher total protein levels by 47%; Depletion of TCA cycle intermediates; Decreased fatty acid content

[62] [66]

[76]

753

The abbreviations are as in Figure 1

**

Higher expression of the Met-rich 10-kDa δ-zein

754

FC, fold-changes compared to WT seeds

755

35