Overexpression of an extraplastidic β-amylase which accumulates in the radish taproot influences the starch content of Arabidopsis thaliana

Abstract

Recent studies have revealed that β-amylases (EC 3.2.1.2) located in plastids participate in endogenous starch degradation. On the other hand, many plants possess a respectable amount of extraplastidic β-amylases. Because of their existence outside of the plastids, it has been believed that extraplastidic β-amylases do not contribute to starch degradation in plants. Here, we investigated the role of the major extraplastidic β-amylase of the radish (Raphanus sativus) (RsBAMY1) in starch metabolism. Amylase activity was elevated in the growing taproot of the radish. During the elevation of amylase activity, the starch content decreased, but the contents of soluble sugars such as maltose, glucose, fructose and sucrose increased. In the radish taproot, RsBAMY1 protein accumulated in the primary cambium and anomalous cambium of the storage parenchyma. The starch granules, however, were found just inside the cambium and adjacent to the anomalous cambium. When the RsBAMY1 gene was overexpressed in Arabidopsis, the plants contained lower starch contents than the wild-type plant. These results suggest that the extraplastidic β-amylase may affect the starch metabolism in some plants.Key words: Cambium, maltose, Raphanus sativus, storage organ.β-Amylase (EC 3.2.1.2) is an amylolytic enzyme which hydrolyses α-1,4 glycosidic linkages at the non-reducing end to β-maltose. Recent molecular genetic studies have provided convincing evidence that plastidic β-amylases are involved in endogenous starch degradation in Arabidopsis leaves (Smith et al. 2005;Fettke et al. 2009;Zeeman et al. 2010). β-Maltose is a primary product of starch degradation in Arabidopsis leaves (Weise et al. 2005). The Arabidopsis genome has nine β-amylase genes. Among them, the four β-amylase proteins (BAMs 1-4) are localized in the plastids, and BAMs 1, 3, and 4 were shown to contribute to starch degradation (Kaplan and Guy 2005;Edner et al. 2007;Fulton et al. 2008;Valerio et al. 2011). The bam3 or bam4 mutant showed a strong starch excess phenotype (Kaplan and Guy 2005;Fulton et al. 2008). The bam1 mutant showed no starch excess phenotype (Kaplan and Guy 2005;Fulton et al. 2008), but a multiple mutant which was produced by crossing the bam1 mutant with the bam3 or bam4 mutant showed a higher starch content compared to bam3 or bam4 (Fulton et al. 2008). The isoforms BAM1 and BAM3 had amylolytic activities, but BAM4 did not (Li et al. 2009), suggesting that BAM4 may modulate the rate of starch degradation as a maltose sensor (Fulton et al. 2008). These results indicate that the plastidic β-amylases directly or indirectly contribute to starch degradation in plants.On the other hand, it is known that a large amount of β-amylase activities, most of which are extraplastidic, are found in many vegetative organs (Beck and Ziegler 1989;Ziegler 1999), such as pea leaves (Ziegler and Beck 1986) and hedge bindweed rhizomes (Van Damme et al. 2001). Despite their abundance, the extraplastidic β-amylases have not been considered to degrade endogenous starch beca...

Transcriptome Profiling of Taproot Reveals Complex Regulatory Networks during Taproot Thickening in Radish (Raphanus sativus L.)

“…Similarly, three transcripts (CL1841.Contig1, CL2019.Contig1 and Unigene1147) encoding beta-amylase, starch degradation related gene, were also up-regulated in three libraries. This finding is similar to that of Takahashi et al ( 2012 ) and Hara et al ( 2009 ) in which beta-amylases activity was elevated in the growing taproot of the radish, where starch content increased, implying that beta-amylases may be storage proteins in plant taproot (Gana et al, 1998 ).…”

Manipulation of the hypocotyl sink activity by reciprocal grafting of two Raphanus sativus varieties: its effects on morphological and physiological traits of source leaves and whole‐plant growth

“…Despite the fact that the detailed mechanism is unclear, it is reported that β ‐amylase is also involved in hypocotyl thickening through degradation of starch and accumulation of soluble sugars (Takahashi et al . ). Thus, for the efficient hypocotyl growth, gene expressions and activities of these proteins must be regulated coordinately.…”

“…It was also reported that the hypocotyl sink activity is related to the activity of β ‐amylase that localizes in the cambia of the hypocotyl cortex (Takahashi et al . ) in several varieties of radish plants (Hara et al . ).…”

Relationship between Major Components and Physicochemical Properties of Radish (Raphanus sativus L.) Combinations for Developing New Cultivars Targeting Chinese Market

“…Seong et al(2016) Table 1과 같다. 기존 연구결과에 따르면 무의 근육에서 측정되는 전분 가수분해 활성의 대 부분은 β-amylase에 의한 것이므로 생성된 환원당 함량을 이용하여 β-amylase 활성을 결정하였다 (Hara et al, 2009b;Takahashi et al, 2012). 본 연구에서 이용한 무의 β-amylase 활성은 최대 169±17 total unit/g에서 최소 41.6±1.3 total unit/g 으로 나타났으며 평균값은 93.2±38.0 total unit/g으로 각 시료에 따라 최대 4배 가량 큰 차이를 보였다.…”

“…또, 맥주 발효에 있어 맥아 생산 시 형성되는 β-amylase는 가장 중요한 발효 영향 인자로 잘 알려져 있다. 무의 경우 β-amylase 활성은 특히 무의 전분 함량 및 대사 과정과 밀접한 관련 이 있다고 보고된 바 있으며(Takahashi et al, 2012) 특정 β-amylase의 활성 정도에 따라 단맛이 우수한 품종을 선별할 수 있다…”