Granule-bound Starch Synthase Gene Research Articles

Evolutionary and expression analysis of starch synthase genes from Tartary buckwheat revealed the potential function of FtGBSSII‐4 and FtGBSSII‐5 in seed amylose biosynthesis

AbstractThe starch synthase has been identified well as a core enzyme in the starch biosynthesis pathway in monocots. However, only a few starch synthase genes (SSs) are known in dicots. Tartary buckwheat (Fagopyrum tataricum Gaertn.) is one of the dicots‐enriched starch in the seeds, but its starch synthesis pathway remains unclear. Here, 15 SS genes were identified in the Tartary buckwheat genome, including 5 granule‐bound starch synthase (GBSS) and 10 soluble starch synthase (SSS) genes. Evolutionary analysis showed that all FtGBSSs belonged to GBSSII, and its isoforms were more abundant than other dicots due to the segmental duplication events. Expression pattern analysis showed two GBSS genes (FtGBSSII‐4 and FtGBSSII‐5) and three SSS genes (FtSSII‐2, FtSSIII‐1, and FtSSIV‐2) were mainly expressed in seeds, indicating that they may be the dominant isoforms for the starch synthesis in Tartary buckwheat. Besides, 23 transcription factor genes out of 1073 were identified from 17 transcription factor families and highly expressed in seeds. Expression correlation analysis suggested that FtNF‐YB2 may be the promising regulators for FtSSs. These results provide new insights and a theoretical basis for understanding starch synthesis and regulation in Tartary buckwheat and other dicots.

Comparative analysis of the transcriptome during single-seed formation of Castanea henryi: regulation of starch metabolism and endogenous hormones

BackgroundIn seed plants, the ovule is the precursor to the seed. The process of ovule development and differentiation is regulated by multiple factors, including starch metabolism and endogenous hormones. Castanea henryi produces nuts with high nutritional value. However, the high proportion of empty buds restricts the commercial use of the tree. Previous studies have shown that the empty bud phenotype is closely related to ovule abortion. If none of the ovules in the ovary expand rapidly and develop in 7–8 weeks after pollination, an empty bud will form. Therefore, we studied the development and molecular mechanisms underlying single seed formation in C. henryi.ResultsWe found that 49 days after pollination (DAP) is a critical period for the formation of fertile and abortive ovules. The morphology and starch distribution of the fertile and abortive ovules differed significantly at 49 DAP. The fertile ovules were smooth and round in appearance, with a large amount of starch. In contrast, abortive ovules were smaller with only a small amount of starch. The embryo sac of the abortive ovule proceeded to develop abnormally, and the entire ovule lacked starch. We identified 37 candidate genes involved in metabolism with potential roles in the regulation of starch levels. Three ADP-glucose pyrophosphorylase (AGPase) genes, one granule-bound starch synthase (GBSS) gene, and two beta-amylase genes could affect starch accumulation. The levels of auxin, cytokinins, gibberellins, and jasmonic acid in fertile ovules were higher than those in abortive ovules. In addition, the levels of endogenous abscisic acid and salicylic acid in abortive ovules were higher than those in fertile ovules of the same age, consistent with the expression patterns of genes related to the synthesis of abscisic and salicylic acid and signal transduction. We identified and mapped the differentially expressed genes associated with hormone synthesis and signal transduction.ConclusionsThese results improve our general understanding of the molecular mechanisms underlying single seed development in C. henryi and the phenomenon of empty buds, providing directions for future research.

Efficient Accumulation of Amylopectin and Its Molecular Mechanism in the Submerged Duckweed Mutant.

Large-scale use of fossil fuels has brought about increasingly serious problems of environmental pollution, development and utilization of renewable energy is one of the effective solutions. Duckweed has the advantages of fast growth, high starch content and no occupation of arable land, so it is a promising starchy energy plant. A new submerged duckweed mutant (sub-1) with abundant starch accumulation was obtained, whose content of amylopectin accounts for 84.04% of the starch granules. Compared with the wild type (Lemna aequinoctialis), the branching degree of starch in sub-1 mutant was significantly increased by 19.6%. Chain length DP 6-12, DP 25-36 and DP > 36 of amylopectin significantly decreased, while chain length DP 13-24 significantly increased. Average chain length of wild-type and sub-1 mutant starches were greater than DP 22. Moreover, the crystal structure and physical properties of starch have changed markedly in sub-1 mutant. For example, the starch crystallinity of sub-1 mutant was only 8.94%, while that of wild-type was 22.3%. Compared with wild type, water solubility of starch was significantly reduced by 29.42%, whereas swelling power significantly increased by 97.07% in sub-1 mutant. In order to further analyze the molecular mechanism of efficient accumulation of amylopectin in sub-1 mutant, metabolome and transcriptome were performed. The results showed that glucose accumulated in sub-1 mutant, then degradation of starch to glucose mainly depends on α-amylase. At night, the down-regulated β-amylase gene resulted in the inhibition of starch degradation. The starch and sucrose metabolism pathways were significantly enriched. Up-regulated expression of SUS, AGPase2, AGPase3, PYG, GPI and GYS provide sufficient substrate for starch synthesis in sub-1 mutant. From the 0H to 16H light treatment, granule-bound starch synthase (GBSS1) gene was inhibited, on the contrary, the starch branching enzyme (SBE) gene was induced. Differential expression of GBSS1 and SBE may be an important reason for the decrease ratio of amylose/amylopectin in sub-1 mutant. Taken together, our results indicated that the sub-1 mutant can accumulate the amylopectin efficiently, potentially through altering the differential expression of AGPase, GBSS1, SBE, and BAM. This study also provides theoretical guidance for creating crop germplasm with high amylopectin by means of synthetic biology in the future.

CRISPR/Cas9-Mediated Mutagenesis of the Granule-Bound Starch Synthase Gene in the Potato Variety Yukon Gold to Obtain Amylose-Free Starch in Tubers.

Potato (Solanum tuberosum L.) is the third most important food crop after rice and wheat. Its tubers are a rich source of dietary carbohydrates in the form of starch, which has many industrial applications. Starch is composed of two polysaccharides, amylose and amylopectin, and their ratios determine different properties and functionalities. Potato varieties with higher amylopectin have many food processing and industrial applications. Using Agrobacterium-mediated transformation, we delivered Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) reagents to potato (variety Yukon Gold) cells to disrupt the granule-bound starch synthase (gbssI) gene with the aim of eliminating the amylose component of starch. Lugol-Iodine staining of the tubers showed a reduction or complete elimination of amylose in some of the edited events. These results were further confirmed by the perchloric acid and enzymatic methods. One event (T2-7) showed mutations in all four gbss alleles and total elimination of amylose from the tubers. Viscosity profiles of the tuber starch from six different knockout events were determined using a Rapid Visco Analyzer (RVA), and the values reflected the amylopectin/amylose ratio. Follow-up studies will focus on eliminating the CRISPR components from the events and on evaluating the potential of clones with various amylose/amylopectin ratios for food processing and other industrial applications.

Transcriptome sequencing and differential expression analysis of seed starch accumulation in Chinese chestnut Metaxenia

BackgroundChestnut seeds are important kinds of edible nuts rich in starch and protein. The characteristics and nutrient contents of chestnut have been found to show obvious metaxenia effects in previous studies. To improve the understanding of the effect of metaxenia on chestnut starch and sucrose metabolism, this study used three varieties of chestnut, ‘Yongfeng 1’, ‘YongRen Zao’ and ‘Yimen 1’, as male parents to pollinate the female parent, ‘Yongfeng 1’, and investigated the mechanisms of starch and sucrose metabolism in three starch accumulation stages (70 (S1), 82 (S2), and 94 (S3) days after pollination, DAP) in chestnut seed kernels.ResultMost carbohydrate metabolism genes were highly expressed in YFF (self-pollinated ‘Yongfeng 1’) in stage S2 and in YFR (‘Yongfeng 1’ × ‘Yongren Zao’) and YFM (‘Yongfeng 1’ × ‘Yimen 1’) in stage S3. In stage S3, hub genes encoding HSF_DNA-binding, ACT, Pkinase, and LIM proteins and four transcription factors were highly expressed, with YFF showing the highest expression, followed by YFR and YFM. In addition, transcriptome analysis of the kernels at 70, 82 and 94 DAP showed that the starch granule-bound starch synthase (EC 2.4.1.242) and ADP-glucose pyrophosphorylase (EC 2.7 .7.27) genes were actively expressed at 94 DAF. Chestnut seeds regulate the accumulation of soluble sugars, reducing sugars and starch by controlling glycosyl transferase and hydrolysis activity during development.ConclusionThese results and resources have important guiding significance for further research on starch and sucrose metabolism and other types of metabolism related to chestnut metaxenia.

Effect of glgB/GASBD fusion gene expression on increased branching degree of potato starch and changes in physicochemical properties of starch

ABSTRACT The objective of this study was to investigate the influence of granule-bound starch-binding domain fusion gene expression on the structure and properties of potato starch. The glgB/GASBD fusion gene and the glgB gene for control were transferred into the potato cultivar Desiree using Agrobacterium-mediated method, which were expressed in potato tubers driven by the potato granule-bound starch synthase gene promoter. The accumulation of glgB/GASBD fusion protein in starch granules was analyzed by Western blot hybridization. A large amount of glgB/GASBD fusion protein was found to accumulate in the starch granules, and the position of the GASBD in the fusion protein (at the N-terminus or at the C-terminus) had no significant effect on the accumulation of the fusion protein in the starch granules. An increase in the degree of branching of the starch results in a specific change in the physical and chemical properties and functional properties of the starch.

Effects of (S)-Carvone and Gibberellin on Sugar Accumulation in Potatoes during Low Temperature Storage.

Potato tubers (Solanum tuberosum L.) are usually stored at low temperature, which can suppress sprouting and control the occurrence of diseases. However, low temperatures lead potatoes to easily suffer from cold-induced sweetening (CIS), which has a negative effect on food processing. The aim of this research was to investigate potential treatments on controlling CIS in potatoes during postharvest storage. “Atlantic” potatoes were treated with gibberellin and (S)-carvone, respectively, and stored at 4 °C for 90 days. The results showed that gibberellin can significantly accelerate sprouting and sugar accumulation by regulating expressions of ADP-glucose pyrophosphorylase (AGPase), granule-bound starch synthase (GBSS), β-amylase (BAM1/2), UDP-glucose pyrophosphorylase (UGPase) and invertase inhibitor (INH1/2) genes. The opposite effects were found in the (S)-carvone treatment group, where CIS was inhibited by modulation of the expressions of GBSS and INH1/2 genes. In summary, gibberellin treatment can promote sugar accumulation while (S)-carvone treatment has some effects on alleviating sugar accumulation. Thus, (S)-carvone can be considered as a potential inhibitor of some of the sugars which are vital in controlling CIS in potatoes. However, the chemical concentration, treatment time, and also the treatment method needs to be optimized before industrial application.

Characterization of a new granule-bound starch synthase gene found in amaranth grains (Amaranthus cruentus L.)

To clarify the mechanism underlying amylose synthesis in the amaranth pericarp, we attempted to identify new GBSS isoforms. A new GBSS-encoding gene (i.e., CrGBSSIb) was isolated from amaranth leaf tissue. The CrGBSSIb gene consists of 4699-bp, including a 1938-bp open reading frame encoding 606 amino acids. A comparison of the cDNA and genomic sequences suggested that CrGBSSIb contains 12 introns and 13 exons. Interestingly, a phylogenetic analysis revealed that the amaranth GBSSIb gene evolved independently from the other GBSSI isoforms within this crop (i.e., intraspecies) and differed from the other plant GBSSII genes. The expression patterns of two GBSS isoforms revealed that CrGBSSIb and CrGBSSI are expressed during the early and late phases of seed development, respectively. Additionally, a high CrGBSSIb transcript level was detected in leaf tissue. This result indicates that CrGBSSI and CrGBSSIb, which affect amylose synthesis in amaranth grains, are active in the perisperm and pericarp, respectively. Therefore, CrGBSSIb encodes an enzyme associated with amylose synthesis. The enzyme may be primarily responsible for amylose metabolism in pericarp tissue.

Waxy and non-waxy barley cultivars exhibit differences in the targeting and catalytic activity of GBSS1a.

Amylose synthesis is strictly associated with activity of granule-bound starch synthase (GBSS) enzymes. Among several crops there are cultivars containing starch types with either little or no amylose known as near-waxy or waxy. This (near) amylose-free phenotype is associated with a single locus (waxy) which has been mapped to GBSS-type genes in different crops. Most waxy varieties are a result of either low or no expression of a GBSS gene. However, there are some waxy cultivars where the GBSS enzymes are expressed normally. For these types, single nucleotide polymorphisms have been hypothesized to represent amino-acid substitutions leading to loss of catalytic activity. We here confirm that the HvGBSSIa enzyme from one such waxy barley variety, CDC_Alamo, has a 90% reduction in catalytic activity. We also engineered plants with expression of transgenic C-terminal green fluorescent protein-tagged HvGBSSIa of both the non-waxy type and of the CDC_Alamo type to monitor their subcellular localization patterns in grain endosperm. HvGBSSIa from non-waxy cultivars was found to localize in discrete concentric spheres strictly within starch granules. In contrast, HvGBSSIa from waxy CDC_Alamo showed deficient starch targeting mostly into unknown subcellular bodies of 0.5-3 µm in size, indicating that the waxy phenotype of CDC_Alamo is associated with deficient targeting of HvGBSSIa into starch granules.

Registration of A/BN641 and RN642 waxy Grain Sorghum Genetic Stocks

Loss-of-function mutations in the granule-bound starch synthase gene result in an endosperm with a waxy appearance and a near absence of starch amylose. Three waxy grain sorghum [Sorghum bicolor (L.) Moench] lines, AN641 (Reg. No. GS-741, PI 672150), BN641 (Reg. No. GS-742, PI 672151), and RN642 (Reg. No. GS-743, PI 672152) were developed jointly by the USDA–ARS and the Agricultural Research Division, Institute of Agriculture and Natural Resources, University of Nebraska, and were released in June 2014. AN641 and BN641 have the waxyb (wxb) allele and are near-isogenic to ‘Wheatland’. RN642 has the waxya (wxa) allele and is near-isogenic to ‘Tx430’. Release of these lines with cytoplasmic male-sterile (A), maintainer (B), and fertility restorer (R) fertility reactions to A1 cytoplasm facilitates the production and evaluation of interallelic (wxb × wxa) waxy and heterowaxy [wxb × wild-type (WT) and WT × wxa] hybrids as a source of low-amylose starch for the ethanol and food industries.

SPECIES IDENTIFICATION AND DIVERSITY IN FRAGARIA L. USING NUCLEAR REGIONS AND UNIVERSAL CHLOROPLAST SSRS

The US National Fragaria collection at the United States Department of Agriculture (USDA), Agricultural Research Service (ARS), National Clonal Germplasm Repository (NCGR) is dynamic through the discovery and collection of new acquisitions and new characteristics that delineate existing species. Previous research using standard Fragaria chloroplast genome regions exhibited limited variation and was unsuccessful at distinguishing between species. The objective of this study was to examine universal chloroplast simple sequence repeats SSRs (cpSSRs) to evaluate diversity in Fragaria species. Four universal cpSSRs (ccmp2, ccmp5, ccmp6, and ccmp7) generated 28 Fragaria haplotypes in ninety four accessions representing 20 Fragaria taxa. Taxa-specific haplotypes were identified for F. nipponica, F. orientalis, F. iinumae and F. nilgerrensis. An octoploid-specific haplotype was observed in cultivated F. x ananassa, F. chiloensis and F. virginiana supporting their close relationship. Genetic diversity using only four universal SSR markers was moderate (0.54, on average) despite the observed homoplasy. Species-specific dehydroascorbate reductase (DHAR) and granule-bound starch synthase (GBSSI-2) gene sequences were also evaluated to identify diploid strawberry species. Marker results and future needs for additional Fragaria-specific primers that may more accurately identify the chloroplast diversity in Fragaria will be discussed.

Bioinformatic analysis of maize granule-bound starch synthase gene

Local alignment of Wx gene and its homolog sequences has been conducted. Phylogenetic dendrogram displaying evolutionary relationship between Poaceae members has been built basing on results of the alignment. Ancient Wx gene transfer from Zea mays to Dimeria lawsonii has been assumed. Primers for the exons 8-10 polymorphic region have been designed. In silico PCR has been conducted.

Identification of Genes Encoding Granule-Bound Starch Synthase Involved in Amylose Metabolism in Banana Fruit

Granule-bound starch synthase (GBSS) is responsible for amylose synthesis, but the role of GBSS genes and their encoded proteins remains poorly understood in banana. In this study, amylose content and GBSS activity gradually increased during development of the banana fruit, and decreased during storage of the mature fruit. GBSS protein in banana starch granules was approximately 55.0 kDa. The protein was up-regulated expression during development while it was down-regulated expression during storage. Six genes, designated as MaGBSSI-1, MaGBSSI-2, MaGBSSI-3, MaGBSSI-4, MaGBSSII-1, and MaGBSSII-2, were cloned and characterized from banana fruit. Among the six genes, the expression pattern of MaGBSSI-3 was the most consistent with the changes in amylose content, GBSS enzyme activity, GBSS protein levels, and the quantity or size of starch granules in banana fruit. These results suggest that MaGBSSI-3 might regulate amylose metabolism by affecting the variation of GBSS levels and the quantity or size of starch granules in banana fruit during development or storage.

Cloning of Granule-Bound Starch Synthase Gene and Construction of Its RNAi Vector in Potato Tuber

There is about 17% starch in potato(Solanum tuberosum L.) tubers.Potato starch granules are composed of two polysaccharides,unbranched amylose(approximately 20% to 25%) and branched amylopectin(approximately 75% to 80%).To develop transgenic potato with high-amylopectin in tubers,we got a cDNA sequence of the potato GBSSI from the total RNA of potato tubers by RT-PCR using specific primers of conserved domain of GenBank Accession Number X58453 sequence.The GBSSI cDNA sequence shared 99.78% similarity with the GBSSI gene in GenBank(accession No.X58453).The full-length of cDNA was 1 824 bp,which contained 607 amino acids,three conserved domains and many important functional sites.The 3D structure of GBSSI was highly similar to that of the glycogen synthase,indicating that GBSSI has a function of starch synthesis.GBSSI similar gene obtained here was granule-bound starch synthase,and its sequence was submitted to GenBank,with the accession number of EU403426.On the basis of a 542 bp RNAi target region from the CDS of GBSSI,the and antisense fragments were amplified and separated by a 237 bp intron to construct the RNA interference expression vector pBI121g-PgABI containing sense gbssA-VP1-ABI3-like protein intron-antisense gbss B regulated by Patatin promoter,which will lay a solid foundation for the study on synthesis of starch and breeding of transgenic potato with high amylopectin content or pure amylopectin.

Cloning of Granule-Bound Starch Synthase Gene and Construction of Its RNAi Vector in Potato Tuber

There is about 17% starch in potato(Solanum tuberosum L.) tubers.Potato starch granules are composed of two polysaccharides,unbranched amylose(approximately 20% to 25%) and branched amylopectin(approximately 75% to 80%).To develop transgenic potato with high-amylopectin in tubers,we got a cDNA sequence of the potato GBSSI from the total RNA of potato tubers by RT-PCR using specific primers of conserved domain of GenBank Accession Number X58453 sequence.The GBSSI cDNA sequence shared 99.78% similarity with the GBSSI gene in GenBank(accession No.X58453).The full-length of cDNA was 1 824 bp,which contained 607 amino acids,three conserved domains and many important functional sites.The 3D structure of GBSSI was highly similar to that of the glycogen synthase,indicating that GBSSI has a function of starch synthesis.GBSSI similar gene obtained here was granule-bound starch synthase,and its sequence was submitted to GenBank,with the accession number of EU403426.On the basis of a 542 bp RNAi target region from the CDS of GBSSI,the sense and antisense fragments were amplified and separated by a 237 bp intron to construct the RNA interference expression vector pBI121g-PgABI containing "sense gbssA-VP1-ABI3-like protein intron-antisense gbss B" regulated by Patatin promoter,which will lay a solid foundation for the study on synthesis of starch and breeding of transgenic potato with high amylopectin content or pure amylopectin.

Identification of two novel waxy alleles and development of their molecular markers in sorghum

High amylopectin grains of waxy sorghum have a high economic value in the food and bioenergy industries because of their increased starch digestibility and higher ethanol conversion rate compared with wild-type sorghum grains. Mutation in the granule-bound starch synthase (GBSS) gene contributes to the waxy phenotype. Two classes of waxy alleles, wx(a) and wx(b), have been characterized previously. In the present work, we identified two novel types of waxy mutations in the sorghum GBSS gene, designated as wx(c) and wx(d). The wx(c) allele has a G deletion at the 5' splicing site of the ninth intron, causing a shift of the 5' cleavage site; in turn, a reading frame shift occurred and resulted in an early translation termination. The wx(d) allele contained a mutation at the 3' splicing site of the 10th intron, which led to a splicing site shift and resulted in the deletion of five amino acids (GTGKK) in the predicted translation product. Furthermore, cleaved amplified polymorphic sequence (CAPS) markers were developed to detect the wx(c) and wx(d) alleles. With these markers, classification of waxy alleles was performed in nearly 100 sorghum accessions from our breeding program. Most waxy sorghum cultivars in China were either wx(a) or wx(c), implying that these two mutations are preferentially maintained during domestic selection in glutinous sorghum production.

Prediction analysis of structure and function of granule-bound starch synthase gene and its encoding protein in cassava (Manihot esculenta Crantz)

Cassava (Manihot esculenta Crantz) is widely considered as one of the most valuable starch source plants with important food material and new energy production. Starch qua the important effectual caloric ingredient were synthesized mainly through a chain of enzymes, in which the committed-step enzymes granule-bound starch synthase (GBSS) were characterized and analyzed with multifarious bioinformatic tools and serves. The results were showed as following: the molecular structures and physicochemical properties of the Megess genes and encoding proteins were calculated; Megbss protein were localized to the cytosol lacking any transmembrane topological structure; the phylogram analysis suggested that the cassava and some crops producing starch were classified into a large groups according to significant functional association and genetic conservation. The secondary structure of the Megbss protein was mainly composed of α-helixes and random coils, and the tertiary structure were modeled successfully, with some key motifs included.Taken together, these results demonstrate that both of Megbss gene and its encoding protein from cassava have the typical molecular structure and function, and the study will lay theoretical foundation for molecular mechanism and genetic regulation researches of starch biosynthesis. Keywords: Cassava, starch, granule-bound starch synthase, bioinformatics.

Diversification of Genes Encoding Granule-Bound Starch Synthase in Monocots and Dicots Is Marked by Multiple Genome-Wide Duplication Events

Starch is one of the major components of cereals, tubers, and fruits. Genes encoding granule-bound starch synthase (GBSS), which is responsible for amylose synthesis, have been extensively studied in cereals but little is known about them in fruits. Due to their low copy gene number, GBSS genes have been used to study plant phylogenetic and evolutionary relationships. In this study, GBSS genes have been isolated and characterized in three fruit trees, including apple, peach, and orange. Moreover, a comprehensive evolutionary study of GBSS genes has also been conducted between both monocots and eudicots. Results have revealed that genomic structures of GBSS genes in plants are conserved, suggesting they all have evolved from a common ancestor. In addition, the GBSS gene in an ancestral angiosperm must have undergone genome duplication ∼251 million years ago (MYA) to generate two families, GBSSI and GBSSII. Both GBSSI and GBSSII are found in monocots; however, GBSSI is absent in eudicots. The ancestral GBSSII must have undergone further divergence when monocots and eudicots split ∼165 MYA. This is consistent with expression profiles of GBSS genes, wherein these profiles are more similar to those of GBSSII in eudicots than to those of GBSSI genes in monocots. In dicots, GBSSII must have undergone further divergence when rosids and asterids split from each other ∼126 MYA. Taken together, these findings suggest that it is GBSSII rather than GBSSI of monocots that have orthologous relationships with GBSS genes of eudicots. Moreover, diversification of GBSS genes is mainly associated with genome-wide duplication events throughout the evolutionary course of history of monocots and eudicots.

An explosive innovation: Phylogenetic relationships of Solanum section Gonatotrichum (Solanaceae)

Solanum is one of the largest plant genera and exhibits a wide range of morphological diversity. Solanum section Gonatotrichum, the focus of this study, is unique within the genus because of its fruits that swell with turgor pressure and explosively dehisce to disperse the seeds. We infer phylogenetic relationships within section Gonatotrichum using DNA sequence data from two nuclear regions (ITS and the granule-bound starch synthase gene [GBSSI or waxy]) and the chloroplast region trnT-F. The resulting phylogenetic trees support the monophyly of the section with the inclusion of Solanum lignescens, a species not previously thought to belong to the group due to the presence of stellate hairs. This inclusion of this species in section Gonatotrichum suggests that the simple, often geniculate hairs of species in the group may represent reduced stellate hairs. The presence of heterantherous flowers appears to be derived in the section, but this character is largely lost in Solanum parcistrigosum.

Plastid-to-Nucleus Retrograde Signals Are Essential for the Expression of Nuclear Starch Biosynthesis Genes during Amyloplast Differentiation in Tobacco BY-2 Cultured Cells

Amyloplasts, a subtype of plastid, are found in nonphotosynthetic tissues responsible for starch synthesis and storage. When tobacco (Nicotiana tabacum) Bright Yellow-2 cells are cultured in the presence of cytokinin instead of auxin, their plastids differentiate from proplastids to amyloplasts. In this program, it is well known that the expression of nucleus-encoded starch biosynthesis genes, such as ADP-Glucose Pyrophosphorylase (AgpS) and Granule-Bound Starch Synthase (GBSS), is specifically induced. In this study, we investigated the roles of plastid gene expression in amyloplast differentiation. Microarray analysis of plastid genes revealed that no specific transcripts were induced in amyloplasts. Nevertheless, amyloplast development accompanied with starch biosynthesis was drastically inhibited in the presence of plastid transcription/translation inhibitors. Surprisingly, the expression of nuclear AgpS and GBSS was significantly repressed by the addition of these inhibitors, suggesting that a plastid-derived signal(s) that reflects normal plastid gene expression was essential for nuclear gene expression. A series of experiments was performed to examine the effects of intermediates and inhibitors of tetrapyrrole biosynthesis, since some of the intermediates have been characterized as candidates for plastid-to-nucleus retrograde signals. Addition of levulinic acid, an inhibitor of tetrapyrrole biosynthesis, resulted in the up-regulation of nuclear AgpS and GBSS gene expression as well as starch accumulation, while the addition of heme showed opposite effects. Thus, these results indicate that plastid transcription and/or translation are required for normal amyloplast differentiation, regulating the expression of specific nuclear genes by unknown signaling mechanisms that can be partly mediated by tetrapyrrole intermediates.