Reduced Starch Accumulation Research Articles

Soybean LEAFY COTYLEDON 1: A Key Target for Genetic Enhancement of Oil Biosynthesis

Soybean is an important oilseed crop that is used as a feed for livestock and has several industrial uses. Lipid biosynthesis and accumulation primarily occur during seed development in plants. This process is regulated by several transcription factors and interconnected biochemical pathways. This study investigated the role of glycine max LEAFY COTYLEDON 1 (GmLEC1) in soybean seed development and the accumulation of storage reserves. The overexpression of GmLEC1 significantly increased the amount of triacylglycerol (TAG) in transgenic Arabidopsis seeds compared to the wild-type and an atlec1 mutant. Similarly, the high expression of GmLEC1 led to a 12% increase in TAG content in transgenic soybean hairy roots compared to the control. GmLEC1 also altered the fatty acid composition in transgenic Arabidopsis seeds and soybean hairy roots. Additionally, the overexpression of GmLEC1 resulted in a reduction in starch accumulation in seeds and vegetative tissues, as well as changes in cotyledon and seed morphology. The cotyledons of the atlec1 mutant displayed abnormal trichome development, and the seeds were smaller and less tolerant to desiccation. A complementation assay in Arabidopsis restored normal cotyledon phenotype and seed size. The main downstream targets of LEC1 are GL2 and WRI1, which were found to participate in fatty acid biosynthesis and trichome formation through the regulation of phytohormones and various transcription factors involved in seed development and maturation. The findings of this study suggest that GmLEC1 controls seed development and regulates the accumulation of seed storage compounds. Furthermore, these results demonstrate that GmLEC1 could be a reliable target for the genetic improvement of oil biosynthesis in soybean.

Overexpression of SlPRE3 alters the plant morphologies in Solanum lycopersicum.

Overexpression of SlPRE3 is detrimental to the photosynthesis and alters plant morphology and root development. SlPRE3 interacts with SlAIF1/SlAIF2/SlPAR1/SlIBH1 to regulate cell expansion. Basic helix-loop-helix (bHLH) transcription factors play crucial roles as regulators in plant growth and development. In this study, we isolated and characterized SlPRE3, an atypical bHLH transcription factor gene. SlPRE3 exhibited predominant expression in the root and moderate expression in the senescent leaves. Comparative analysis with the wild type revealed significant differences in plant morphology in the 35S:SlPRE3 lines. These differences included increased internode length, rolling leaves with reduced chlorophyll accumulation, and elongated yet fewer adventitious roots. Additionally, 35S:SlPRE3 lines displayed elevated levels of GA3 (gibberellin A3) and reduced starch accumulation. Furthermore, utilizing the Y2H (Yeast two-hybrid) and the BiFC (Bimolecular Fluorescent Complimentary) techniques, we identified physical interactions between SlPRE3 and SlAIF1 (ATBS1-interacting factor 1)/SlAIF2 (ATBS1-interacting factor 2)/SlPAR1 (PHYTOCHROME RAPIDLY REGULATED 1)/SlIBH1 (ILI1-binding bHLH 1). RNA-seq analysis of root tissues revealed significant alterations in transcript levels of genes involved in gibberellin metabolism and signal transduction, cell expansion, and root development. In summary, our study sheds light on the crucial regulatory role of SlPRE3 in determining plant morphology and root development.

Long-Term Soil Drought Limits Starch Accumulation by Altering Sucrose Transport and Starch Synthesis in Sweet Potato Tuberous Root.

In this study, the influences of long-term soil drought with three levels [soil-relative water content (SRWC) (75 ± 5)%, as the control; SRWC (55 ± 5)%, mild drought; SRWC (45 ± 5)%, severe drought] were investigated on sucrose-starch metabolism in sweet potato tuberous roots (TRs) by pot experiment. Compared to the control, drought stress increased soluble sugar and sucrose content by 4-60% and 9-75%, respectively, but reduced starch accumulation by 30-66% through decreasing the starch accumulate rate in TRs. In the drought-treated TRs, the inhibition of sucrose decomposition was attributed to the reduced activities of acid invertase (AI) and alkaline invertase (AKI) and the IbA-INV3 expression, rather than sucrose synthase (SuSy), consequently leading to the increased sucrose content in TRs. In addition, starch synthesis was inhibited mainly by reducing ADP-glucose pyrophosphorylase (AGPase), granular starch synthase (GBSS) and starch branching enzyme (SBE) activities in TRs under drought stress, and AGPase was the rate-limiting enzyme. Furthermore, soil drought remarkably up-regulated the IbSWEET11, IbSWEET605, and IbSUT4 expressions in Jishu 26 TRs, while it down-regulated or had no significant differences in Xushu 32 and Ningzishu 1 TRs. These results suggested that the sucrose-loading capability in Jishu 26 TRs were stronger than that in Xushu 32 and Ningzishu 1 TRs. Moreover, IbA-INV3, IbAGPS1, IbAGPS2, IbGBSSI and IbSBEII play important roles in different drought-tolerant cultivars under drought stress.

A Reduced Starch Level in Plants at Early Stages of Infection by Viruses Can Be Considered a Broad-Range Indicator of Virus Presence.

Simple SummaryIn this report, we describe a potential broad-range indicator of virus presence in plants. This method is based on the observation that starch content in leaves is generally reduced upon virus infection at the early stages of infection. This method was also used to effectively detect the infection of three latent viruses in propagative apple materials. Hence, the plant with the low virus accumulation, latent infection or at early stage of infection can be indicated by assessing starch accumulation levels. The genes encoding key enzymes involved in starch biosynthesis are transcriptionally downregulated by virus infection, suggesting that plant virus infection generally affects cellular metabolic pathways.The diagnosis of virus infection can facilitate the effective control of plant viral diseases. To date, serological and molecular methods for the detection of virus infection have been widely used, but these methods have disadvantages if applied for broad-range and large-scale detection. Here, we investigated the effect of infection of several different plant RNA and DNA viruses such as cucumber mosaic virus (CMV), tobacco mosaic virus (TMV), potato virus X (PVX), potato virus Y (PVY) and apple geminivirus on starch content in leaves of Nicotiana benthamiana. Analysis showed that virus infection at an early stage was generally associated with a reduction in starch accumulation. Notably, a reduction in starch accumulation was readily apparent even with a very low virus accumulation detected by RT-PCR. Furthermore, we also observed that the infection of three latent viruses in propagative apple materials was associated with a reduction in starch accumulation levels. Analysis of transcriptional expression showed that some genes encoding enzymes involved in starch biosynthesis were downregulated at the early stage of CMV, TMV, PVX and PVY infections, suggesting that virus infection interferes with starch biosynthesis in plants. Our findings suggest that assessing starch accumulation levels potentially serve as a broad-range indicator for the presence of virus infection.

Characterizing the impact of high temperature during grain filling on phytohormone levels, enzyme activity and metabolic profiles of an early indica rice variety.

Global warming results in high temperature stress (HTS), which presents severe challenges worldwide for modern agricultural production and will have significant impacts on the yield and quality of crops. Accumulation of photosynthetic products, activity of enzymes involved in sucrose-starch metabolism, phytohormone levels and metabolic profiling using LC-MS were analysed in the flag leaves and/or developing grains subjected to HTS during the grain-filling stage of an indica rice. HTS induced significant yield loss and reduced the grain quality, with lower amylose content. HTS reduced photosynthetic product accumulation in flag leaves and reduced starch accumulation in developing grains, compared to growth under normal temperatures. The activity of enzymes related to sucrose-starch metabolism were dis-regulated in developing grains grown under high temperature (HT). Moreover, phytohormone homeostasis in flag leaves and developing grains was also dramatically disturbed by HT. Metabolic profiling detected many metabolites with remarkably different relative fold abundances at different time points in the developing grain at HT versus normal temperatures, these metabolites were enriched in several HTS response pathways. The change in phytohormone ratio and auxin level might be associated with the reduction in photosynthetic products and their translocation, and ultimately with reduced starch accumulation in the developing grain. The detected metabolites might have different roles in response to the HTS in developing grain at different development stages. These results provide a theoretical reference and basis for future rice production towards higher quality and yield when grown under HTS.

RGB1 Regulates Grain Development and Starch Accumulation Through Its Effect on OsYUC11-Mediated Auxin Biosynthesis in Rice Endosperm Cells.

RGB1, a subunit of heterotrimeric G protein, plays important roles in regulating grain size and weight of rice. However, the molecular mechanisms underlying controlling grain filling process by G protein are still largely unclear. In the present study, we show that RGB1 controls not only the grain size but also the grain filling process. Knock-down of RGB1 significantly delayed grain development and reduced starch accumulation and grain weight, which was closely related to the delayed and the lower expression of genes encoding sucrose metabolism and starch biosynthesis related enzymes during grain filling stage. Suppression of RGB1 expression also resulted in the lower auxin content in grains, which was correlated with the lower expression of OsNF-YB1 and OsYUC11 during grain filling stage. Further biochemical evidence showed that OsYUC11 expression was under control of OsNF-YB1 by its interaction with promoter of OsYUC11. Taken together, we propose that RGB1 controls rice grain development and grain filling process by changing auxin homeostasis in endosperm cells. OsNF-YB1, which acts as a key downstream effector of RGB1, interacts directly with the promoter of OsYUC11 and stimulates the OsYUC11 expression, thereby regulating auxin biosynthesis and starch accumulation and grain size.

The promoted lateral root 1 (plr1) mutation is involved in reduced basal shoot starch accumulation and increased root sugars for enhanced lateral root growth in rice

Lateral roots (LRs) are indispensable for plant growth, adaptability and productivity. We previously reported a rice mutant, exhibiting a high density of thick and long LRs (L-type LRs) with long parental roots and herein referred to as promoted lateral root1 (plr1). In this study, we describe that the mutant exhibited decreased basal shoot starch accumulation, suggesting that carbohydrates might regulate the mutant root phenotype. Further analysis revealed that plr1 mutation gene regulated reduced starch accumulation resulting in increased root sugars for the regulation of promoted LR development. This was supported by the exogenous glucose application that promoted L-type LRs. Moreover, nitrogen (N) application was found to reduce basal shoot starch accumulation in both plr1 mutant and wild-type seedlings, which was due to the repressed expression of starch biosynthesis genes. However, unlike the wild-type that responded to N treatment only at seedling stage, the plr1 mutant regulated LR development under low to increasing N levels, both at seedling and higher growth stages. These results suggest that plr1 mutation gene is involved in reduced basal shoot starch accumulation and increased root sugar level for the promotion of L-type LR development, and thus would be very useful in improving rice root architecture.

Arabidopsis thaliana FAR-RED ELONGATED HYPOCOTYLS3 (FHY3) and FAR-RED-IMPAIRED RESPONSE1 (FAR1) modulate starch synthesis in response to light and sugar.

In living organisms, daily light/dark cycles profoundly affect cellular processes. In plants, optimal growth and development, and adaptation to daily light-dark cycles, require starch synthesis and turnover. However, the underlying molecular mechanisms coordinating daily starch metabolism remain poorly understood. To explore the roles of Arabidopsis thaliana light signal transduction proteins FAR-RED ELONGATED HYPOCOTYLS3 (FHY3) and FAR-RED-IMPAIRED RESPONSE1 (FAR1) in starch metabolism, the contents of starch and water-soluble polysaccharides, and the structure of starch granules were investigated in fhy3, far1 and fhy3 far1 mutant plants. Disruption of FHY3 or FAR1 reduced starch accumulation and altered starch granule structure in the fhy3-4, far1-2, and fhy3-4 far1-2 mutant plants. Furthermore, molecular and genetic evidence revealed that the gene encoding the starch-debranching enzyme ISOAMYLASE2 (ISA2) is a direct target of FHY3 and FAR1, and functions in light-induced starch synthesis. Our data establish the first molecular link between light signal transduction and starch synthesis, suggesting that the light-signaling proteins FHY3 and FAR1 influence starch synthesis and starch granule formation through transcriptional activation of ISA2.

Comparative in vitro analyses of recombinant maize starch synthases SSI, SSIIa, and SSIII reveal direct regulatory interactions and thermosensitivity

Starch synthases SSI, SSII, and SSIII function in assembling the amylopectin component of starch, but their specific roles and means of coordination are not fully understood. Genetic analyses indicate regulatory interactions among SS classes, and physical interactions among them are known. The N terminal extension of cereal SSIII, comprising up to 1200 residues beyond the catalytic domain, is responsible at least in part for these interactions. Recombinant maize SSI, SSIIa, and full-length or truncated SSIII, were tested for functional interactions regarding enzymatic activity. Amino-terminal truncated SSIII exhibited reduced activity compared to full-length enzyme, and addition of the N terminus to the truncated protein stimulated catalytic activity. SSIII and SSI displayed a negative interaction that reduced total activity in a reconstituted system. These data demonstrate that SSIII is both a catalytic and regulatory factor. SSIII activity was reduced by approximately 50% after brief incubation at 45 °C, suggesting a role in reduced starch accumulation during growth in high temperatures. Buffer effects were tested to address a current debate regarding the SS mechanism. Glucan stimulated the SSIIa and SSIII reaction rate regardless of the buffer system, supporting the accepted mechanism in which glucosyl units are added to exogenous primer substrates.

Starch Granule Re-Structuring by Starch Branching Enzyme and Glucan Water Dikinase Modulation Affects Caryopsis Physiology and Metabolism.

Starch is of fundamental importance for plant development and reproduction and its optimized molecular assembly is potentially necessary for correct starch metabolism. Re-structuring of starch granules in-planta can therefore potentially affect plant metabolism. Modulation of granule micro-structure was achieved by decreasing starch branching and increasing starch-bound phosphate content in the barley caryopsis starch by RNAi suppression of all three Starch Branching Enzyme (SBE) isoforms or overexpression of potato Glucan Water Dikinase (GWD). The resulting lines displayed Amylose-Only (AO) and Hyper-Phosphorylated (HP) starch chemotypes, respectively. We studied the influence of these alterations on primary metabolism, grain composition, starch structural features and starch granule morphology over caryopsis development at 10, 20 and 30 days after pollination (DAP) and at grain maturity. While HP showed relatively little effect, AO showed significant reduction in starch accumulation with re-direction to protein and β-glucan (BG) accumulation. Metabolite profiling indicated significantly higher sugar accumulation in AO, with re-partitioning of carbon to accumulate amino acids, and interestingly it also had high levels of some important stress-related metabolites and potentially protective metabolites, possibly to elude deleterious effects. Investigations on starch molecular structure revealed significant increase in starch phosphate and amylose content in HP and AO respectively with obvious differences in starch granule morphology at maturity. The results demonstrate that decreasing the storage starch branching resulted in metabolic adjustments and re-directions, tuning to evade deleterious effects on caryopsis physiology and plant performance while only little effect was evident by increasing starch-bound phosphate as a result of overexpressing GWD.

The Omp85-type outer membrane protein p36 of Arabidopsis thaliana evolved by recent gene duplication.

Proteins of the Omp85 family are involved in the insertion of β-barrel shaped outer membrane proteins in bacteria and mitochondria, and-at least-in the transfer of preproteins across the chloroplast outer envelope. In general these proteins consist of up to five N-terminal "polypeptide transport associated" (POTRA) domains and a C-terminal, membrane embedded β-barrel domain. In Arabidopsis thaliana two plastidic gene families coding for Omp85-like proteins exist, namely the Toc75-III and the Toc75-V/Oep80 sub-family. The latter is composed of three genes, of which two do not contain POTRA domains. These are annotated as P39 and P36. However, P36 resulted from a very recent gene duplication of P39 and appears to be specific to Arabidopsis thaliana. Furthermore, we show that P39 is specifically expressed in vein tissues, while P36 is expressed at early and late developmental stages. T-DNA insertion in P36 causes a mild phenotype with reduced starch accumulation in chloroplasts of sepals pointing towards a yet to be described plastid function.

Effect of Drought Stress During Flowering Stage on Starch Accumulation and Starch Synthesis Enzymes in Sorghum Grains

Starch content is a key factor affecting sorghum grain quality. The research of sorghum grain starch accumulation and the related synthesis enzyme activities has great significance for understanding the mechanisms of starch metabolisms. The differences between a high and a low starch content sorghum hybrids (Tieza 17 and Liaoza 11, respectively) in grain starch accumulation and the related synthesis enzyme activities were assessed following imposition of water stress during flowering stage. The total starch, amylase and amylopectin accumulation all decreased at the mid-late stage of grain filling under drought stress during flowering stage. The maximum and mean accumulation rates also decreased. During grain filling, soluble starch synthase (SSS), granule-bound starch synthase (GBSS), starch branching enzyme (SBE), and starch debranching enzymes (DBE) activities were all affected, though differently. Drought stress reduced starch accumulation in a larger extent for Tieza 17 than Liaoza 11. Drought stress during flowing stage reduced starch synthesis enzyme activities, thus reducing starch accumulation in grains, and the differences between starch components were also demonstrated under drought stress.

Rice chalky ring formation caused by temporal reduction in starch biosynthesis during osmotic adjustment under foehn-induced dry wind.

Foehn-like extreme hot and dry wind conditions (34°C, >2.5 kPa vapor pressure deficit, and 7 m s−1) strongly affect grain quality in rice (Oryza sativa L.). This is a current concern because of the increasing frequency and intensity of combined heat and water-deficit stress under climate change. Foehn-induced dry wind conditions during the grain-filling stage increase ring-shaped chalkiness as a result of spatiotemporal reduction in starch accumulation in the endosperm, but kernel growth is sometimes maintained by osmotic adjustment. Here, we assess the effects of dry wind on chalky ring formation in environmentally controlled growth chambers. Our results showed that hot and dry wind conditions that lasted for >24 h dramatically increased chalky ring formation. Hot and dry wind conditions temporarily reduced panicle water potential to –0.65 MPa; however, kernel growth was maintained by osmotic adjustment at control levels with increased transport of assimilate to the growing kernels. Dynamic tracer analysis with a nano-electrospray-ionization Orbitrap mass spectrometer and quantitative polymerase chain reaction analysis revealed that starch degradation was negligible in the short-term treatment. Overall expression of starch synthesis-related genes was found to be down-regulated at moderately low water potential. Because the events observed at low water potential preceded the packing of starch granules in cells, we concluded that reduced rates of starch biosynthesis play a central role in the events of cellular metabolism that are altered at osmotic adjustment, which leads to chalky ring formation under short-term hot and dry wind conditions.

The sink-specific plastidic phosphate transporter PHT4;2 influences starch accumulation and leaf size in Arabidopsis.

Nonphotosynthetic plastids are important sites for the biosynthesis of starch, fatty acids, and amino acids. The uptake and subsequent use of cytosolic ATP to fuel these and other anabolic processes would lead to the accumulation of inorganic phosphate (Pi) if not balanced by a Pi export activity. However, the identity of the transporter(s) responsible for Pi export is unclear. The plastid-localized Pi transporter PHT4;2 of Arabidopsis (Arabidopsis thaliana) is expressed in multiple sink organs but is nearly restricted to roots during vegetative growth. We identified and used pht4;2 null mutants to confirm that PHT4;2 contributes to Pi transport in isolated root plastids. Starch accumulation was limited in pht4;2 roots, which is consistent with the inhibition of starch synthesis by excess Pi as a result of a defect in Pi export. Reduced starch accumulation in leaves and altered expression patterns for starch synthesis genes and other plastid transporter genes suggest metabolic adaptation to the defect in roots. Moreover, pht4;2 rosettes, but not roots, were significantly larger than those of the wild type, with 40% greater leaf area and twice the biomass when plants were grown with a short (8-h) photoperiod. Increased cell proliferation accounted for the larger leaf size and biomass, as no changes were detected in mature cell size, specific leaf area, or relative photosynthetic electron transport activity. These data suggest novel signaling between roots and leaves that contributes to the regulation of leaf size.

Does leaf photosynthesis adapt to CO2-enriched environments? An experiment on plants originating from three natural CO2 springs.

Atmospheric CO2 elevation may act as a selective agent, which consequently may alter plant traits in the future. We investigated the adaptation to high CO2 using transplant experiments with plants originating from natural CO2 springs and from respective control sites. We tested three hypotheses for adaptation to high-CO2 conditions: a higher photosynthetic nitrogen use efficiency (PNUE); a higher photosynthetic water use efficiency (WUE); and a higher capacity for carbohydrate transport from leaves. Although elevated growth CO2 enhanced both PNUE and WUE, there was no genotypic improvement in PNUE. However, some spring plants had a higher WUE, as a result of a significant reduction in stomatal conductance, and also a lower starch concentration. Higher natural variation (assessed by the coefficient of variation) within populations in WUE and starch concentration, compared with PNUE, might be responsible for the observed population differentiation. These results support the concept that atmospheric CO2 elevation can act as a selective agent on some plant traits in natural plant communities. Reduced stomatal conductance and reduced starch accumulation are highlighted for possible adaptation to high CO2.

The reduction of starch accumulation in transgenic sugarcane cell suspension culture lines

Starch only occurs in small amounts in sugarcane, but is, nevertheless an unwanted product because it reduces the amount of sucrose that can be crystallized from molasses. In an attempt to reduce the starch content of sugarcane, the activities of ADP-glucose pyrophosphorylase (AGPase) and beta-amylase were manipulated using transgenic approaches. Transformation vectors to reduce AGPase activity and to increase plastidial beta-amylase activity were constructed and used for the transformation of sugarcane calli. The results of the manipulations were analyzed in suspension cultures. AGPase activity was reduced down to between 14 and 54% of the wild-type control. This led to a reduction in starch concentration down to 38% of the levels of the wild-type control. beta-Amylase activity was increased in the transgenic lines by 1.5-2 times that of the wild-type control. This increase in activity led to a reduction in starch amounts by 90% compared to wild-type control cells. In both experiments, the changes in starch concentrations could be correlated with the change in enzyme activity. There were no significant effects on sucrose concentrations in either experiment, indicating that these approaches might be useful to engineer regenerated sugarcane for optimized sucrose production.

Mutation of the Plastidial α-Glucan Phosphorylase Gene in Rice Affects the Synthesis and Structure of Starch in the Endosperm

Plastidial phosphorylase (Pho1) accounts for approximately 96% of the total phosphorylase activity in developing rice (Oryza sativa) seeds. From mutant stocks induced by N-methyl-N-nitrosourea treatment, we identified plants with mutations in the Pho1 gene that are deficient in Pho1. Strikingly, the size of mature seeds and the starch content in these mutants showed considerable variation, ranging from shrunken to pseudonormal. The loss of Pho1 caused smaller starch granules to accumulate and modified the amylopectin structure. Variation in the morphological and biochemical phenotype of individual seeds was common to all 15 pho1-independent homozygous mutant lines studied, indicating that this phenotype was caused solely by the genetic defect. The phenotype of the pho1 mutation was temperature dependent. While the mutant plants grown at 30 degrees C produced mainly plump seeds at maturity, most of the seeds from plants grown at 20 degrees C were shrunken, with a significant proportion showing severe reduction in starch accumulation. These results strongly suggest that Pho1 plays a crucial role in starch biosynthesis in rice endosperm at low temperatures and that one or more other factors can complement the function of Pho1 at high temperatures.

Exposing pepper plants to high day temperatures prevents the adverse low night temperature symptoms

Exposing pepper (Capsicum annuum) plants to extremely high day temperatures (HDT) (day/night temperatures of 36 ± 2/10 ± 2°C), obtained by keeping the greenhouse closed during the day to exploit solar heating, prevented the development of low night temperature (LNT) symptoms. Plants of cultivars Fiesta and Selica grown under LNTs (10 ± 2°C) and moderate day temperatures (25 ± 2°C) during winter exhibited retarded growth, reduced leaf numbers, and deformed fruits with few or no seeds. LNT caused a reduction in the number and quality of pollen grains: the reduction in pollen quality was associated with reduced starch accumulation in pollen grains at 3 days before anthesis (DBA) and a decrease of more than two‐fold in total soluble sugars in the mature pollen grains. This inhibitory effect was associated with more than 50% reduction in the enzymatic activities of the cell wall‐bound and soluble acid invertases that catalyze the hydrolysis of incoming sucrose molecules. All these symptoms were prevented by HDT treatment which matched the vegetative and reproductive performance of the plants to those of plants grown under optimal night temperature (ONT) conditions (day/night temperatures of 23 ± 2/18 ± 2°C). HDT also prevented the inhibitory effect of LNT on enzymatic activities of both invertases in pollen at 5 DBA and brought about the accumulation of high levels of starch in pollen at 3 DBA. The results presented could support the development of a novel procedure for producing greenhouse crops with minimum or even with no fuel consumption for heating during the winter nights in regions with bright and sunny days.

The effect of high temperatures on the expression and activity of sucrose-cleaving enzymes during tomato (Lycopersicon esculentum) anther development

SummaryExposure to high temperatures causes reduced yields in tomato (Lycopersicon esculentum), mainly by affecting male gametophyte development. The effect of heat stress on pollen characteristics is associated with changes in carbohydrate metabolism in the developing anthers. The expression and activities of sucrose-cleaving enzymes and the amounts of starch and soluble sugars were measured in tomato (inbred line ‘NC 8288’) anthers at four stages of development, under ‘normal’ controlled (day/night temperatures of 26°/18°C) and high-temperature (day/night temperatures of 32°/26°C) conditions. Heat stress was found to cause a reduction in cell wall-bound acid invertase activity in the anthers of flower buds 5 d before anthesis (DBA), which correlated with reduced starch accumulation at 3 DBA, and an induction of the activities of sucrose synthase and soluble acid invertase in maturing anthers. The heat stress-induced increases in soluble acid invertase and sucrose synthase activities close to anthesis were found to correlate with a three-fold increase in the hexose:sucrose ratio in mature anthers. The heat-stress conditions applied altered the gene expression profiles of the enzymes tested, decreasing the steady-state level of the mRNA coding for cell wall-bound acid invertase at 5 DBA, increasing expression of the soluble acid invertase gene at 5 DBA, and decreasing expression of the sucrose synthase gene at 5 and 3 DBA. These data indicate that sucrose-cleaving enzymes in tomato anthers respond, at both the mRNA and enzyme activity levels, to high-temperature conditions, that the response is dependent upon the stage of flower development, and that it may involve post-transcriptional control.

The T-DNA Oncogene A4-orf8 from Agrobacterium rhizogenes A4 Induces Abnormal Growth in Tobacco

The related orf8 and iaaM T-DNA genes from Agrobacterium are each composed of two distinct parts. The 5' parts (called Norf8 or NiaaM) encode a 200-amino-acid (aa) sequence with homology to various T-DNA oncoproteins such as RolB, RolC, and 6b. The 3' parts (Corf8 or CiaaM) encode a 550-aa sequence with homology to IaaM proteins from Pseudomonas and Pantoea spp. Whereas iaaM genes encode flavin adenine dinucleotide (FAD)-dependent tryptophan 2-monooxygenases that catalyze the synthesis of indole-3-acetamide (IAM), A4-orf8 from Agrobacterium rhizogenes A4 does not. Plants expressing a 2x35S-A4-Norf8 construct accumulate soluble sugars and starch. We now have regenerated plants that express the full-size 2x35S-A4-orf8 and the truncated 2x35S-A4-Corf8 gene. 2x35S-A4-Corf8 plants accumulate starch and show reduced growth like 2x35S-A4-Norf8 plants but, in addition, display a novel set of characteristic growth modifications. These consist of leaf hypertrophy and hyperplasia (blisters); thick, dark-green leaves; thick stems; and swollen midveins. Mutations in the putative FAD-binding site of A4-Orf8 did not affect the blister syndrome. Plants expressing 2x35S-A4-Corf8 had a normal phenotype but contained less starch and soluble sugars than did wild-type plants. When 2x35S-A4-Corf8 plants were crossed to starch-accumulating 2x35S-A4-Norf8 plants with reduced growth, A4-Corf8 partially restored growth and reduced starch accumulation. A4-Corf8xA4-Norf8 crosses did not lead to the blister syndrome, suggesting that this requires physical linkage of the A4-NOrf8 and A4-COrf8 sequences.