Molecular and functional characterization of a family of amino acid transporters from Arabidopsis

More than 50 distinct amino acid transporter genes have been identified in the genome of Arabidopsis, indicating that transport of amino acids across membranes is a highly complex feature in plants. Based on sequence similarity, these transporters can be divided into two major superfamilies: the amino acid transporter family and the amino acid polyamine choline transporter family. Currently, mainly transporters of the amino acid transporter family have been characterized. Here, a molecular and functional characterization of amino acid polyamine choline transporters is presented, namely the cationic amino acid transporter (CAT) subfamily. CAT5 functions as a high-affinity, basic amino acid transporter at the plasma membrane. Uptake of toxic amino acid analogs implies that neutral or acidic amino acids are preferentially transported by CAT3, CAT6, and CAT8. The expression profiles suggest that CAT5 may function in reuptake of leaking amino acids at the leaf margin, while CAT8 is expressed in young and rapidly dividing tissues such as young leaves and root apical meristem. CAT2 is localized to the tonoplast in transformed Arabidopsis protoplasts and thus may encode the long-sought vacuolar amino acid transporter.     

Altered growth and improved resistance of Arabidopsis against Pseudomonas syringae by overexpression of the basic amino acid transporter AtCAT1

Amino acid transporters in plants are crucial for distributing amino acids between plant organs and cellular compartments. The H⁺‐coupled plasma membrane transporter CAT1 (cationic amino acid transporter 1) facilitates the high‐affinity uptake of basic amino acids. The uptake of lysine (Lys) via the roots was not altered in loss‐of‐function mutants, in accordance with the minor expression of CAT1 in roots, but plants ectopically overexpressing CAT1 incorporated Lys at higher rates. Exogenous Lys inhibited the primary root of Arabidopsis, whereas lateral roots were stimulated. These effects were augmented by the presence or absence of CAT1. Furthermore, the total biomass of soil‐grown plants ectopically overexpressing CAT1 was reduced and the time to flowering was accelerated. These effects were accompanied by only minor changes in the overall amino acid profile. Interestingly, CAT1 belongs to a specific small cluster of nitrogen‐containing metabolite transporter genes that are rapidly up‐regulated upon infection with Pseudomonas syringae and that may participate in the systemic response of plants to pathogen attack. The overexpression of CAT1 indeed enhanced the resistance to the hemibiotrophic bacterial pathogen P. syringae via a constitutively activated salicylic acid (SA) pathway, which is consistent with the developmental defects and the resistance phenotype.     

γ-氨基丁酸 (GABA) 毛叶茶品质成分分析

以毛叶茶为研究对象,通过真空厌氧处理将其制作成γ 氨基丁酸(GABA)毛叶茶,探求毛叶茶经厌氧处理后的品质成分变化。结果表明：(1)厌氧处理后的毛叶茶,其GABA含量显著提高,达到GABA茶标准。游离氨基酸、黄酮和生物碱含量也显著升高,但茶多酚和水浸出物含量降低。同时,真空处理还能促进儿茶素的转化。简单儿茶素含量呈降低趋势,ECG和CG含量显著提高,EGCG、GCG含量及酯型儿茶素总量却先增加后降低,最终总量与对照样无明显差异。(2) 毛叶茶中除含有一般的蛋白质氨基酸外,还含有普通茶树品种所特有的特征氨基酸Thea,以及微量的GABA。游离氨基酸中含量较高的有Thea、Glu、Asp,较低的是Met、Cit、α ABA、Tau、Gly。Cysthi和EOHNH2是GABA毛叶茶中特有氨基酸。在真空厌氧条件下,GABA毛叶茶的游离氨基酸由于蛋白质发生降解而总量增加。其中P Ser、Thr、Ser、Asn、Pro、Gly、Cit、α ABA、Val、Cysthi、Ile、Leu、Tyr、Phe、GABA、Trp、Lys、His含量上升,Asp、Glu和α AAA含量均降低,而Ala 和Arg含量却呈现先增后降的趋势,Thea、Cys、Met等游离氨基酸含量在真空处理后无明显变化。     

可可茶经栽培后化学成分的变化及其与传统茶的比较分析

野生茶树可可茶(Camellia Ptilophylla Chang)由于其芽叶中的嘌呤生物碱主要为可可碱,因而不同于传统茶叶。通过化学筛选,在纯种无性苗建立的可可茶基地上,随机进行单株取样,对可可茶的水浸出物、游离氨基酸、水溶性糖、茶多酚、儿茶素类、花青素、嘌呤生物碱等成分进行了检测。将这些结果与野生可可茶相关成份进行比较,发现可可茶经人工栽培后,含优势可可碱的特点保持不变,游离氨基酸总量和儿茶素类含量得到了明显的提高。进一步与传统茶叶比较后,得出两者之间的最大差异是可可茶含可可碱,不含咖啡碱;传统茶叶含咖啡碱为主,同时伴生相当于0．5～1％咖啡碱量的可可碱。     

烟草磷转运蛋白基因NtPHT2;1的克隆和表达分析

植物对磷酸盐的吸收与利用主要依靠磷转运蛋白,其中PHT2家族编码的低亲和磷转运蛋白主要负责植物在正常供磷条件下磷酸盐的吸收、转运与再利用。为了探究低亲和磷转运蛋白基因NtPHT2;1在烟草转运磷酸盐中的作用和表达模式,本研究以普通烟草K326的cDNA为模板,克隆得到NtPHT2;1,对该基因进行生物信息学分析和蛋白质的亚细胞定位,并通过荧光定量PCR技术对该基因在低磷等非生物胁迫下的基因表达模式进行分析。结果表明：（1）NtPHT2;1基因的全长为1 764 bp,编码587个氨基酸。（2）亚细胞定位结果表明,NtPHT2;1蛋白定位于叶绿体上。（3）同源性比对发现,NtPHT2;1蛋白与辣椒CaPHT2;1蛋白的同源性最高达到91.00%。（4）启动子分析表明,NtPHT2;1启动子含有参与调控植物衰老、逆境胁迫相关的顺式作用元件。（5）组织表达模式分析表明,NtPHT2;1在叶片中的表达量最高,新叶中的表达量比老叶中的高;在低磷诱导条件下,该基因的表达量与正常条件相比差异不显著。（6）不同非生物胁迫下的表达模式表明,在盐胁迫和干旱胁迫下,该基因的表达量显著降低。研究认为,NtPHT2;1基因主要是负责烟株正常生长发育条件下磷酸盐的转运与利用。     

Theanine transporters identified in tea plants (Camellia sinensis L.)

Theanine, a unique non‐proteinogenic amino acid, is an important component of tea, as it confers the umami taste and relaxation effect of tea as a beverage. Theanine is primarily synthesized in tea roots and is subsequently transported to young shoots, which are harvested for tea production. Currently, the mechanism for theanine transport in the tea plant remains unknown. Here, by screening a yeast mutant library, followed by functional analyses, we identified the glutamine permease, GNP1 as a specific transporter for theanine in yeast. Although there is no GNP1 homolog in the tea plant, we assessed the theanine transport ability of nine tea plant amino acid permease (AAP) family members, with six exhibiting transport activity. We further determined that CsAAP1, CsAAP2, CsAAP4, CsAAP5, CsAAP6, and CsAAP8 exhibited moderate theanine affinities and transport was H⁺‐dependent. The tissue‐specific expression of these six CsAAPs in leaves, vascular tissues, and the root suggested their broad roles in theanine loading and unloading from the vascular system, and in targeting to sink tissues. Furthermore, expression of these CsAAPs was shown to be seasonally regulated, coincident with theanine transport within the tea plant. Finally, CsAAP1 expression in the root was highly correlated with root‐to‐bud transport of theanine, in seven tea plant cultivars. Taken together, these findings support the hypothesis that members of the CsAAP family transport theanine and participate in its root‐to‐shoot delivery in the tea plant.     

茶树冷诱导基因RAV的克隆与表达特性分析

对利用cDNA-AFLP技术所获得的茶树低温诱导差异表达片段TDF,通过RACE方法获得含完整编码区序列的茶树RAV基因cDNA克隆,其开放阅读框编码361个氨基酸,包含两个保守的结构域AP2和B3,与多种植物RAV蛋白具有高度同源性。qRT-PCR分析表明,茶树RAV基因受低温、乙烯、NaCl等上调表达,最大表达量分别是诱导前的5.8、10.0和1.9倍。在成熟叶片、芽、嫩茎中RAV基因表达量相近,花蕾和嫩根中表达较低,而在种子中不表达。推测该基因在组织中的表达受到严格控制以及在响应非生物胁迫中发挥重要作用。     

8个茶树品种生化成分分析及抗性成分的初步鉴定

【目的】筛选和鉴定茶树中与假眼小绿叶蝉Empoasca vitis G?the相关的抗性成分。【方法】采用氨基酸分析仪和超高效液相色谱法分别分析8个茶树品种的氨基酸及其组分、咖啡碱和茶多酚及其组分的含量。采用人工饲料喂饲法对可能的抗性物质进行初步鉴定。【结果】通过对假眼小绿叶蝉抗性水平差异较大的茶树品种的30多种生化物质的分析,筛选出抗、感茶树品种间差异较大的物质,包括茶多酚、天冬氨酸、γ-氨基丁酸、绿原酸和茶氨酸。分别将其作为可变因子设置不同浓度梯度,加入到人工饲料饲养假眼小绿叶蝉,统计5种化学成份不同浓度人工饲料上假眼小绿叶蝉的成活率,初步确定了这5种化学成份对假眼小绿叶蝉成活率的影响。其中,γ-氨基丁酸可能是茶树抗虫物质之一。【结论】为阐明茶树抗叶蝉的化学机理奠定基础。     

Predicted Functional Shifts Due to Type of Soil Microbiome and Watering of Two Wild Plants in Western Region of Saudi Arabia

The present study aimed to predict differential enrichment of pathways and compounds in the rhizosphere microbiomes of the two wild plants (Abutilon fruticosum and Nitrosalsola vermiculata) and to predict functional shifts in microbiomes due to water. Amplicon sequencing of 16S rRNA region V3–V4 was done and gene-based microbial compositions were enrolled in PICRUSt to predict enriched pathways and compounds. The results indicated that “ABC transporters” and “Quorum sensing” pathways are among the highest enriched pathways in rhizosphere microbiomes of the two wild plants compared with those of the bulk soil microbiomes. The highest enriched compounds in soil microbiomes of the two wild plants included five proteins and three enzymes participating in one or more KEGG pathways. Six of these eight compounds showed higher predicted enrichment in rhizosphere soil microbiomes, while only one, namely phosphate transport system substrate-binding protein, showed higher enrichment in the surrounding bulk soil microbiomes. In terms of differentially enriched compounds due to watering, only the dual-specific aspartyl-tRNA (Asn)/glutamyl-tRNA (Gln) amidotransferase subunit A showed higher enrichment in rhizosphere soil of the two wild plants after 24 h of watering. Two of the highly enriched compounds namely branched-chain amino acid transport system ATP-binding protein and branched-chain amino acid transport system substrate-binding protein, are encoded by genes stimulated by the plant’s GABA that participates in conferring biotic and abiotic stresses in plants and improves the plant’s growth performance. The 3-Oxoacyl-[ACP] reductase, a member of the short-chain alcohol dehydrogenase/ reductase (SDR) superfamily, participates in fatty acids elongation cycles and contributes to plant-microbe symbiotic relationships, while enoyl-CoA hydratase has a reverse action as it participates in “Fatty acid degradation” pathway. The methyl-accepting chemotaxis protein is an environmental signal that sense “Bacterial chemotaxis” pathway to help establishing symbiosis with plant roots by recruiting/colonizing of microbial partners (symbionts) to plant rhizosphere. This information justifies the high enrichment of compounds in plant rhizosphere. The dual-specific aspartyl-tRNA (Asn)/glutamyl-tRNA (Gln) amidotransferase subunit A contributes to the plant ability to respond to watering as it participates in attaching the correct amino acid during translation to its cognate tRNA species, while hydrolyzing incorrectly attached amino acid. These two actions reduce the influence of oxidative stress in generating misfolded proteins and in reducing fidelity of translation.     

Functional analysis of <Emphasis Type="Italic">OsPUT1</Emphasis>, a rice polyamine uptake transporter

Polyamines are nitrogenous compounds found in all eukaryotic and prokaryotic cells and absolutely essential for cell viability. In plants, they regulate several growth and developmental processes and the levels of polyamines are also correlated with the plant responses to various biotic and abiotic stresses. In plant cells, polyamines are synthesized in plastids and cytosol. This biosynthetic compartmentation indicates that the specific transporters are essential to transport polyamines between the cellular compartments. In the present study, a phylogenetic analysis was used to identify candidate polyamine transporters in rice. A full-length cDNA rice clone AK068055 was heterologously expressed in the Saccharomyces cerevisiae spermidine uptake mutant, agp2∆. Radiological uptake and competitive inhibition studies with putrescine indicated that rice gene encodes a protein that functioned as a spermidine-preferential transporter. In competition experiments with several amino acids at 25-fold higher levels than spermidine, only methionine, asparagine, and glutamine were effective in reducing uptake of spermidine to 60% of control rates. Based on those observations, this rice gene was named polyamine uptake transporter 1 (OsPUT1). Tissue-specific expression of OsPUT1 by semiquantitative RT-PCR showed that the gene was expressed in all tissues except seeds and roots. Transient expression assays in onion epidermal cells and rice protoplasts failed to localize to a cellular compartment. The characterization of the first plant polyamine transporter sets the stage for a systems approach that can be used to build a model to fully define how the biosynthesis, degradation, and transport of polyamines in plants mediate developmental and biotic responses.     

Characterization of novel small RNAs from tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis> L.)

Small RNAs play important roles in plant development, metabolism, signal transduction and responses to biotic and abiotic stresses by affecting gene expression. Tea (Camellia sinensis L.) is an important commercial crop in the world. To understand the regulatory mechanisms involving small RNAs in tea metabolism, we constructed a small RNA (sRNA) library from its tea drink manufacturing tissue part i.e. topmost two leaves and a bud. For the first time, we isolated and cloned six novel small RNAs candidates from tea. These were predicted to target 67 genes responsible for various important plant functions. Isolated small RNAs were validated through expression analysis in young leaf and old leaf during non-dormant and dormant growth phases of tea. Results suggest the probable role of isolated small RNAs in development and seasonal variations of tea.     

Characterization of ammonium and nitrate uptake and assimilation in roots of tea plants

It has been pointed out that tea (Camellia sinensis (L.) O. Kuntze) prefers ammonium (NH ₄ ⁺ ) over nitrate (NO ₃ ^? ) as an inorganic nitrogen (N) source. ¹⁵N studies were conducted using hydroponically grown tea plants to clarify the characteristics of uptake and assimilation of NH ₄ ⁺ and NO ₃ ^? by tea roots. The total ¹⁵N was detected, and kinetic parameters were calculated after feeding ¹⁵NH ₄ ⁺ or ¹⁵NO ₃ ^? to tea plants. The process of N assimilation was studied by monitoring the dynamic ¹⁵N abundance in the free amino acids of tea plant roots by GC-MS. Tea plants supplied with ¹⁵NH ₄ ⁺ absorbed significantly more ¹⁵N than those supplied with ¹⁵NO ₃ ^? . The kinetics of ¹⁵NH ₄ ⁺ and ¹⁵NO ₃ ^? influx into tea plants followed a classic biphasic pattern, demonstrating the action of a high affinity transport system (HATS) and a low affinity transport system (LATS). The V _max value for NH ₄ ⁺ uptake was 54.5 nmol/(g dry wt min), which was higher than that observed for NO ₃ ^? (39.3 nmol/(g dry wt min)). K_M estimates were approximately 0.06 mM for NH ₄ ⁺ and 0.16 mM for NO ₃ ^? , indicating a higher rate of NH ₄ ⁺ absorption by tea plant roots. Tea plants fed with ¹⁵NH ₄ ⁺ accumulated larger amounts of assimilated N, especially glutamine (Gln), compared with those fed with ¹⁵NO ₃ ^? . Gln, Glu, theanine (Thea), Ser, and Asp were the main free amino acids that were labeled with ¹⁵N under both conditions. The rate of N assimilation into Thea in the roots of NO ₃ ^? -supplied tea plants was quicker than in NH ₄ ⁺ -supplied tea plants. NO ₃ ^? uptake by roots, rather than reduction or transport within the plant, seems to be the main factor limiting the growth of tea plants supplied with NO ₃ ^? as the sole N source. The NH ₄ ⁺ absorbed by tea plants directly, as well as that produced by NO ₃ ^? reduction, was assimilated through the glutamine synthetase-glutamine oxoglutarate aminotransferase pathway in tea plant roots. The ¹⁵N labeling experiments showed that there was no direct relationship between the Thea synthesis and the preference of tea plants for NH ₄ ⁺ .     

GABA/BABA priming: a means for enhancing abiotic stress tolerance potential of plants with less energy investments on defence cache

Abiotic stress is one of the major factors limiting plant growth and yield globally. Though substantial progress has been made in breeding and genetic manipulation of plants to enhance abiotic stress tolerance, the task remains as a challenge even today. Investigations on the priming activity of various chemicals in plants for enhancing abiotic stress tolerance have been undertaken over the past few years. Priming with γ-amino butyric acid (GABA) and β-amino butyric acid (BABA) gains greater attention, because priming with these non-protein amino acids equips the plants to resist abiotic stresses effectively without suffering costly energy investments in operating defence mechanisms. It is well documented that the protective effect of non-protein amino acids like BABA and GABA on plants is due to a potentiation of natural defence mechanisms against abiotic stresses but at the same time not activating the complete defence arsenal before the stress exposure. The exact mode of action of priming with GABA/BABA in plants is still a puzzle, though their importance as signaling molecules during stress is undoubtful. The better understanding of molecular, physiological, and ecological aspects of GABA/BABA priming might lead to the emergence of this technique as a successful strategy for enhancing the abiotic stress(es) tolerance potential of plants in the field, without compromising much on productivity.     

Characterization of L-arginine transport in adrenal cells: effect of ACTH

Nitric oxide synthesis depends on the availability of its precursor L-arginine, which could be regulated by the presence of a specific uptake system. In the present report, the characterization of the L-arginine transport system in mouse adrenal Y1 cells was performed. L-arginine transport was mediated by the cationic/neutral amino acid transport system y+L and the cationic amino acid transporter (CAT) y+ in Y1 cells. These Na+-independent transporters were identified by their selectivity for neutral amino acids in both the presence and absence of Na+ and by the effect of N-ethylmaleimide. Transport data correlated to expression of genes encoding for CAT-1, CAT-2, CD-98, and y+LAT-2. A similar expression profile was detected in rat adrenal zona fasciculata. In addition, cationic amino acid uptake in Y1 cells was upregulated by ACTH and/or cAMP with a concomitant increase in nitric oxide (NO) production.     

Reduced amino acid content in transgenic potato tubers due to antisense inhibition of the leaf H+/amino acid symporter StAAP1

Transport processes across the plasma membrane of leaf vascular tissue are essential for transport and distribution of assimilates. In potato, leaves are the predominant sites for nitrate reduction and amino acid biosynthesis. From there, assimilated amino acids are exported through the phloem to supply tubers with organic nitrogen. To study the role of amino acid transporters in long-distance transport and allocation of organic nitrogen in potato plants, a gene encoding a functional, leaf-expressed amino acid permease StAAP1 was isolated. Similar to the sucrose transporter SUT1, StAAP1 expression was induced during the sink-to-source transition, indicating a role in phloem loading. To test the role of StAAP1, expression was inhibited by an antisense approach. Transgenic plants with reduced StAAP1 expression were phenotypically indistinguishable from wild type, as were photosynthetic capacity and tuber yield. However, tubers from antisense StAAP1 plants showed up to 50% reduction in free amino acid contents. In comparison, starch content was not affected or tended to increase relative to wild type. The reduction in all amino acids except aspartate in the antisense plants is consistent with the properties of amino acid permeases (AAPs) found in heterologous systems. The results demonstrate an important role for StAAP1 in long-distance transport of amino acids and highlight the importance of plasma membrane transport for nutrient distribution in plants.