Study of AtSUS2 localization in seeds reveals a strong association with plastids

Sucrose synthase (SUS) is a key enzyme in sucrose metabolism. This enzyme catalyzes the reversible conversion of sucrose and UDP to UDP-glucose and fructose. In the Arabidopsis SUS gene family (six members), SUS2 is strongly and specifically expressed in Arabidopsis seeds during the maturation phase. Using specific antibodies, we have shown that SUS2 is localized in the embryo, endosperm and seed coat with differential patterns. During the maturation phase, the SUS2 protein seems to be mainly co-localized with plastids in the embryo. This novel finding is discussed in relation to the role of this enzyme in storage organs.     

No evidence for the occurrence of substrate inhibition of Arabidopsis thaliana sucrose synthase-1 (AtSUS1) by fructose and UDP-glucose

Sucrose synthase (SuSy) catalyzes the reversible conversion of sucrose and NDP into the corresponding nucleotide-sugars and fructose. The Arabidopsis genome possesses six SUS genes (AtSUS1–6) that code for proteins with SuSy activity. As a first step to investigate optimum fructose and UDP-glucose (UDPG) concentrations necessary to measure maximum sucrose-producing SuSy activity in crude extracts of Arabidopsis, in this work we performed kinetic analyses of recombinant AtSUS1 in two steps: (1) SuSy reaction at pH 7.5, and (2) chromatographic measurement of sucrose produced in step 1. These analyses revealed a typical Michaelis-Menten behavior with respect to both UDPG and fructose, with Km values of 50 μM and 25 mM, respectively. Unlike earlier studies showing the occurrence of substrate inhibition of UDP-producing AtSUS1 by fructose and UDP-glucose, these analyses also revealed no substrate inhibition of AtSUS1 at any UDPG and fructose concentration. By including 200 mM fructose and 1 mM UDPG in the SuSy reaction assay mixture, we found that sucrose-producing SuSy activity in leaves and stems of Arabidopsis were exceedingly higher than previously reported activities. Furthermore, we found that SuSy activities in organs of the sus1/sus2/sus3/sus4 mutant were ca. 80–90% of those found in WT plants.     

Isolation of the gene encoding Carrot leafy cotyledon1 and expression analysis during somatic and zygotic embryogenesis.

The Arabidopsis thaliana LEC1 gene regulates embryo morphology and seed maturation. For a better understanding of its function, we isolated a carrot (Daucus carota L. cv. US-Harumakigosun) counterpart of this gene, C-LEC1, from a cDNA library of carrot somatic embryos, since carrot is a better model plant for preparing large quantities of somatic embryos at the same developmental stage. The predicted amino acid sequence of C-LEC1 is similar to that of LEC1 and contains regions that are conserved in the heme-activated protein 3 (HAP3) subunit of plants, animals and microorganisms. C-LEC1 expression was detected in embryogenic cells, somatic embryos, and developing seeds. In situ hybridization analysis revealed C-LEC1 expression in the peripheral region of the embryos but not in the endosperm. Expression of C-LEC1 driven by Arabidopsis LEC1 promoter was able to complement the defects of the Arabidopsis lec1-1 mutant. These results suggest that C-LEC1 is a functional homolog of Arabidopsis LEC1, an important regulator of zygotic and somatic embryo development.     

Arabidopsis sucrose synthase 2 and 3 modulate metabolic homeostasis and direct carbon towards starch synthesis in developing seeds

Two genes encoding sucrose synthase (SUS), namely SUS2 (At5g49190) and SUS3 (At4g02280), are strongly and differentially expressed in Arabidopsis seed. Detailed biochemical analysis was carried out in developing seeds 9–21 days after flowering (DAF) of wild type and two knockouts. SUS2 and SUS3 are not redundant genes since single knockouts show a phenotype in developing seeds. The mutants had 30–50% less SUS activity and therefore accumulated 40% more sucrose and 50% less fructose at 15 DAF. This did not affect the hexose-P pool, but led to 30–70% less starch in embryo and seed coat. Lipids were 55% higher in both mutants at 9–15 DAF. It seems that sucrolysis via SUS is not required for oil or protein synthesis but rather for channeling carbon toward ADP-glucose and starch in seeds. Metabolite profiling with GC–TOF revealed specific downstream changes in primary metabolism as a consequence of signaling or regulatory fine-tuning. While sucrose increased, hexoses and specific amino acids decreased reciprocally. There was a developmental shift regarding an earlier timing of dry weight accumulation, germinative maturity, oil deposition, sugar levels, transient starch buildup, and protein storage. Nevertheless, final seed size and composition were unaltered due to an earlier cessation of growth, thus giving rise to an apparent silent phenotype of mature mutant seeds. We conclude that SUS is important for metabolite homeostasis and timing of seed development, and propose that an altered sucrose/hexose ratio can modify carbon partitioning and the pattern of storage compounds in Arabidopsis.     

AtGA3ox2, a key gene responsible for bioactive gibberellin biosynthesis, is regulated during embryogenesis by LEAFY COTYLEDON2 and FUSCA3 in Arabidopsis

Embryonic regulators LEC2 (LEAFY COTYLEDON2) and FUS3 (FUSCA3) are involved in multiple aspects of Arabidopsis (Arabidopsis thaliana) seed development, including repression of leaf traits and premature germination and activation of seed storage protein genes. In this study, we show that gibberellin (GA) hormone biosynthesis is regulated by LEC2 and FUS3 pathways. The level of bioactive GAs is increased in immature seeds of lec2 and fus3 mutants relative to wild-type level. In addition, we show that the formation of ectopic trichome cells on lec2 and fus3 embryos is a GA-dependent process as in true leaves, suggesting that the GA pathway is misactivated in embryonic mutants. We next demonstrate that the GA-biosynthesis gene AtGA3ox2, which encodes the key enzyme AtGA3ox2 that catalyzes the conversion of inactive to bioactive GAs, is ectopically activated in embryos of the two mutants. Interestingly, both beta-glucuronidase reporter gene expression and in situ hybridization indicate that FUS3 represses AtGA3ox2 expression mainly in epidermal cells of embryo axis, which is distinct from AtGA3ox2 pattern at germination. Finally, we show that the FUS3 protein physically interacts with two RY elements (CATGCATG) present in the AtGA3ox2 promoter. This work suggests that GA biosynthesis is directly controlled by embryonic regulators during Arabidopsis embryonic development.     

LEAFY COTYLEDON 2 activation is sufficient to trigger the accumulation of oil and seed specific mRNAs in Arabidopsis leaves

LEAFY COTYLEDON 2 (LEC2) is a key regulator of seed maturation in Arabidopsis. To unravel some of its complex pleiotropic functions, analyses were performed with transgenic plants expressing an inducible LEC2:GR protein. The chimeric protein is functional and can complement lec2 mutation. Interestingly, the induction of LEC2 leads to the accumulation of storage oil in leaves. In addition, short-term induction and use of translation inhibitors allowed to demonstrate that LEC2 can directly trigger the accumulation of seed specific mRNAs. Consistent with these results, the expression of three other major seed regulators namely, LEC1, FUS3, and ABI3 were also induced by LEC2 activation.     

A network of local and redundant gene regulation governs Arabidopsis seed maturation

In Arabidopsis thaliana, four major regulators (ABSCISIC ACID INSENSITIVE3 [ABI3], FUSCA3 [FUS3], LEAFY COTYLEDON1 [LEC1], and LEC2) control most aspects of seed maturation, such as accumulation of storage compounds, cotyledon identity, acquisition of desiccation tolerance, and dormancy. The molecular basis for complex genetic interactions among these regulators is poorly understood. By analyzing ABI3 and FUS3 expression in various single, double, and triple maturation mutants, we have identified multiple regulatory links among all four genes. We found that one of the major roles of LEC2 was to upregulate FUS3 and ABI3. The lec2 mutation is responsible for a dramatic decrease in ABI3 and FUS3 expression, and most lec2 phenotypes can be rescued by ABI3 or FUS3 constitutive expression. In addition, ABI3 and FUS3 positively regulate themselves and each other, thereby forming feedback loops essential for their sustained and uniform expression in the embryo. Finally, LEC1 also positively regulates ABI3 and FUS3 in the cotyledons. Most of the genetic controls discovered were found to be local and redundant, explaining why they had previously been overlooked. This works establishes a genetic framework for seed maturation, organizing the key regulators of this process into a hierarchical network. In addition, it offers a molecular explanation for the puzzling variable features of lec2 mutant embryos.     

蔗糖合酶基因AtSUS3干涉后对拟南芥角果发育的影响

蔗糖合酶(SuSy)是植物蔗糖代谢关键酶之一,该研究利用反向遗传学手段,采用RNAi技术抑制拟南芥中AtSUS3基因的表达,测定纯系转基因植株的抽苔率,并对酶活性、糖含量等指标以及糖代谢相关基因的表达进行了检测,探讨SuSy在植物发育中的作用。结果显示:(1)转基因拟南芥的抽苔平均早于野生型植株2～3d,且优先3～4d完成抽苔。(2)开花后生长天数对角果蔗糖和葡萄糖含量有显著影响,而对果糖含量影响不显著;开花后5d时,野生型株系的葡萄糖含量显著高于转基因株系SUS3-2,至15d时,两种转基因株系葡萄糖含量均显著低于野生型株系。(3)开花后生长天数对SuSy、SPS、INV的活性均有显著影响,随开花时间延长,野生型株系SuSy活性显著低于转基因株系,而SPS和INV则相反。(4)AtSUS3基因沉默对其他糖代谢基因有不同程度的影响,开花后5d时,转基因植株的角果中AtCesA1、AtCesA7和AtCINV1的表达量较野生型都有所增加;开花后15d时,转基因植株的角果中AtCesA1、AtCesA7的表达量较野生型高,而AtCINV、AtCwINV的表达量比野生型低。研究表明,拟南芥AtSUS3基因沉默后,在正常生长条件下未造成植株发育异常,同时还可能通过同源家族中其他SuSy的表达水平增加,促进了该酶及糖代谢相关基因整体水平的增加,有助于角果成熟。     

Two sesquiterpene synthases are responsible for the complex mixture of sesquiterpenes emitted from Arabidopsis flowers

Despite the fact that Arabidopsis is largely self-pollinating, its flowers emit a complex mixture of terpene volatiles consisting predominantly of a large group of over 20 sesquiterpenes. Here we report that only two terpene synthases, encoded by the florally expressed genes At5g23960 and At5g44630, are responsible for the formation of virtually all sesquiterpenes found in the Arabidopsis floral volatile blend. Two independent mutant lines with T-DNA insertions in the previously identified At5g23960 gene lacked the emission of three sesquiterpenes, including the main sesquiterpene volatile (E)-beta-caryophyllene, confirming the previous in vitro functional assignment for this gene. Flowers of a mutant line carrying a T-DNA insertion in gene At5g44630 emitted these three sesquiterpenes, but did not emit any of the remaining sesquiterpene volatiles. An At5g44630 cDNA was expressed in Escherichia coli and the produced protein catalyzed the conversion of farnesyl diphosphate into over 15 sesquiterpenes in similar proportions to those found in the floral volatile blend. At5g23960 and At5g44630 promoter-beta-glucuronidase (GUS) fusion experiments demonstrated that both genes are expressed in several parts of the Arabidopsis flower, with strong At5g23960 promoter-GUS activity in the stigma and strong expression of At5g44630 in intrafloral nectaries. Given the previously reported antimicrobial activity of terpenes, their production in stigmas and nectaries may serve to inhibit microbial infection at these vulnerable sites. A survey of 37 Arabidopsis thaliana ecotypes revealed quantitative, but almost no qualitative, variations of floral monoterpene and sesquiterpene emissions suggesting that floral terpene volatiles must play some significant role in the life of the Arabidopsis plant.