ZmEREB92 plays a negative role in seed germination by regulating ethylene signaling and starch mobilization in maize.

Rapid and uniform seed germination is required for modern cropping system. Thus, it is important to optimize germination performance through breeding strategies in maize, in which identification for key regulators is needed. Here, we characterized an AP2/ERF transcription factor, ZmEREB92, as a negative regulator of seed germination in maize. Enhanced germination in ereb92 mutants is contributed by elevated ethylene signaling and starch degradation. Consistently, an ethylene signaling gene ZmEIL7 and an α-amylase gene ZmAMYa2 are identified as direct targets repressed by ZmEREB92. OsERF74, the rice ortholog of ZmEREB92, shows conserved function in negatively regulating seed germination in rice. Importantly, this orthologous gene pair is likely experienced convergently selection during maize and rice domestication. Besides, mutation of ZmEREB92 and OsERF74 both lead to enhanced germination under cold condition, suggesting their regulation on seed germination might be coupled with temperature sensitivity. Collectively, our findings uncovered the ZmEREB92-mediated regulatory mechanism of seed germination in maize and provide breeding targets for maize and rice to optimize seed germination performance towards changing climates.

An L-type lectin receptor-like kinase promotes starch accumulation during rice pollen maturation.

Starch accumulation is key for the maturity of rice pollen grains; however, the regulatory mechanism underlying this process remains unknown. Here, we have isolated a male-sterile rice mutant, abnormal pollen 1 (ap1), which produces nonviable pollen grains with defective starch accumulation. Functional analysis revealed that AP1 encodes an active L-type lectin receptor-like kinase (L-LecRLK). AP1 is localized to the plasma membrane and its transcript is highly accumulated in pollen during the starch synthesis phase. RNA-seq and phosphoproteomic analysis revealed that the expression/phosphorylation levels of numerous genes/proteins involved in starch and sucrose metabolism pathway were significantly altered in the mutant pollen, including a known rice UDP-glucose pyrophosphorylase (OsUGP2). We further found that AP1 physically interacts with OsUGP2 to elevate its enzymatic activity, likely through targeted phosphorylation. These findings revealed a novel role of L-LecRLK in controlling pollen maturity via modulating sucrose and starch metabolism.

FLOURY ENDOSPERM24, a heat shock protein 101 (HSP101), is required for starch biosynthesis and endosperm development in rice.

Endosperm is the main storage organ in cereal grain and determines grain yield and quality. The molecular mechanisms of heat shock proteins in regulating starch biosynthesis and endosperm development remain obscure. Here, we report a rice floury endosperm mutant flo24 that develops abnormal starch grains in the central starchy endosperm cells. Map-based cloning and complementation test showed that FLO24 encodes a heat shock protein HSP101, which is localized in plastids. The mutated protein FLO24T296I dramatically lost its ability to hydrolyze ATP and to rescue the thermotolerance defects of the yeast hsp104 mutant. The flo24 mutant develops more severe floury endosperm when grown under high-temperature conditions than normal conditions. And the FLO24 protein was dramatically induced at high temperature. FLO24 physically interacts with several key enzymes required for starch biosynthesis, including AGPL1, AGPL3 and PHO1. Combined biochemical and genetic evidence suggests that FLO24 acts cooperatively with HSP70cp-2 to regulate starch biosynthesis and endosperm development in rice. Our results reveal that FLO24 acts as an important regulator of endosperm development, which might function in maintaining the activities of enzymes involved in starch biosynthesis in rice.

The endosperm-specific transcription factor TaNAC019 regulates glutenin and starch accumulation and its elite allele improves wheat grain quality.

In wheat (Triticum aestivum L.), breeding efforts have focused intensively on improving grain yield and quality. For quality, the content and composition of seed storage proteins (SSPs) determine the elasticity of wheat dough and flour processing quality. Moreover, starch levels in seeds are associated with yield. However, little is known about the mechanisms that coordinate SSP and starch accumulation in wheat. In this study, we explored the role of the endosperm-specific NAC transcription factor TaNAC019 in coordinating SSP and starch accumulation. TaNAC019 binds to the promoters of TaGlu-1 loci, encoding high molecular weight glutenin (HMW-GS), and of starch metabolism genes. Triple knock-out mutants of all three TaNAC019 homoeologs exhibited reduced transcript levels for all SSP types and genes involved in starch metabolism, leading to lower gluten and starch contents, and in flour processing quality parameters. TaNAC019 directly activated the expression of HMW-GS genes by binding to a specific motif in their promoters and interacting with the TaGlu-1 regulator TaGAMyb. TaNAC019 also indirectly regulated the expression of TaSPA, an ortholog of maize Opaque2 that activates SSP accumulation. Therefore, TaNAC019 regulation of starch- and SSP-related genes has key roles in wheat grain quality. Finally, we identified an elite allele (TaNAC019-BI) associated with flour processing quality, providing a candidate gene for breeding wheat with improved quality.

Physiological and gene expression response mechanisms of dehydration process in Cinnamomum cassia (L.) J. Presl seeds.

BACKGROUND: A critical factor in the storability of recalcitrant seeds is their moisture content (MC), but its effect on the viability of Cinnamomum cassia (L.) J. Presl (C. cassia) seeds is not fully understood. METHODS AND RESULTS: Measured the germination rate, starch and soluble sugar content, and transcriptome of 8 seed samples with different MC obtained by low-temperature drying method. It was found that the germination rate was significantly negatively correlated with MC. The lethal MC was around 15.6%. During the dehydration process, there was a significant increase in the content of soluble sugars and starch. Transcriptome analysis was performed on CK, W3, W6 showed a total of 62.78 Gb of clean data. Among the 30,228 Unigenes, 28,195 were successfully annotated. In the three comparative groups (CK and W3, CK and W6, W3 and W6), 6,842, 7,640, and 11,628 differentially expressed genes (DEGs) were obtained, respectively. These DEGs were found to be involved in a variety of metabolic pathways, including carbon metabolism, amino acid biosynthesis, starch and sucrose metabolism, glycolysis/gluconeogenesis, and nucleotide and amino sugar metabolism. A total of 1,416 common genes were identified among all three comparison groups. Furthermore, among all the DEGs, a total of 71 transcription factor families were identified, with the C2H2 transcription factor family having the highest number of genes. CONCLUSIONS: This ground-breaking study sheds light on the physiological response and gene expression profiles of C. cassia seeds after undergoing dehydration treatment, which will provide valuable insights for further research and understanding of this process.

QTL Mapping and Candidate Gene Analysis for Starch-Related Traits in Tartary Buckwheat (Fagopyrum tataricum (L.) Gaertn).

Starch is the main component that determines the yield and quality of Tartary buckwheat. As a quantitative trait, using quantitative trait locus (QTL) mapping to excavate genes associated with starch-related traits is crucial for understanding the genetic mechanisms involved in starch synthesis and molecular breeding of Tartary buckwheat varieties with high-quality starch. Employing a recombinant inbred line population as research material, this study used QTL mapping to investigate the amylose, amylopectin, and total starch contents across four distinct environments. The results identified a total of 20 QTLs spanning six chromosomes, which explained 4.07% to 14.41% of the phenotypic variation. One major QTL cluster containing three stable QTLs governing both amylose and amylopectin content, qClu-4-1, was identified and located in the physical interval of 39.85-43.34 Mbp on chromosome Ft4. Within this cluster, we predicted 239 candidate genes and analyzed their SNP/InDel mutations, expression patterns, and enriched KEGG pathways. Ultimately, five key candidate genes, namely FtPinG0004897100.01, FtPinG0002636200.01, FtPinG0009329200.01, FtPinG0007371600.01, and FtPinG0005109900.01, were highlighted, which are potentially involved in starch synthesis and regulation, paving the way for further investigative studies. This study, for the first time, utilized QTL mapping to detect major QTLs controlling amylose, amylopectin, and total starch contents in Tartary buckwheat. The QTLs and candidate genes would provide valuable insights into the genetic mechanisms underlying starch synthesis and improving starch-related traits of Tartary buckwheat.

Plasma membrane-localized SEM1 protein mediates sugar movement to sink rice tissues.

The translocation of photosynthate carbohydrates, such as sucrose, is critical for plant growth and crop yield. Previous studies have revealed that sugar transporters, plasmodesmata and sieve plates act as important controllers in sucrose loading into and unloading from phloem in the vascular system. However, other pivotal steps for the regulation of sucrose movement remain largely elusive. In this study, characterization of two starch excesses in mesophyll (sem) mutants and dye and sucrose export assays were performed to provide insights into the regulatory networks that drive source-sink relations in rice. Map-based cloning identified two allelic mutations in a gene encoding a GLUCAN SYNTHASE-LIKE (GSL) protein, thus indicating a role for SEM1 in callose biosynthesis. Subcellular localization in rice showed that SEM1 localized to the plasma membrane. In situ expression analysis and GUS staining showed that SEM1 was mainly expressed in vascular phloem cells. Reduced sucrose transport was found in the sem1-1/1-2 mutant, which led to excessive starch accumulation in source leaves and inhibited photosynthesis. Paraffin section and transmission electron microscopy experiments revealed that less-developed vascular cells (VCs) in sem1-1/1-2 potentially disturbed sugar movement. Moreover, dye and sugar trafficking experiments revealed that aberrant VC development was the main reason for the pleiotropic phenotype of sem1-1/1-2. In total, efficient sucrose loading into the phloem benefits from an optional number of VCs with a large vacuole that could act as a buffer holding tank for sucrose passing from the vascular bundle sheath.

Genetic diversity assessment and genome-wide association study reveal candidate genes associated with component traits in sweet potato (Ipomoea batatas (L.) Lam).

The Korean sweet potatoes were bred by various cultivars introduced from Japanese, American, Porto Rico, China, and Burundi. This issue enriched their genetic diversity but also resulted in a mixture of cultivars. For genotyping, we collected and sequenced 66 sweet potato germplasms from different localities around Korea, including 36 modern cultivars, 5 local cultivars, and 25 foreign cultivars. This identified 447.6 million trimmed reads and 324.8 million mapping reads and provided 39,424 single nucleotide polymorphisms (SNPs) markers. Phylogenetic clustering and population structure analysis distinctly classified these germplasms into 5 genetic groups, group 1, group 2, group 3, group 4, and group 5, containing 20, 15, 10, 7, and 14 accessions, respectively. Sixty-three significant SNPs were selected by genome-wide association for sugar composition-related traits (fructose, glucose, and total sugars), total starch, amylose content, and total carotenoid of the storage root. A total of 37 candidate genes encompassing these significant SNPs were identified, among which, 7 genes were annotated to involve in sugar and starch metabolism, including galactose metabolism (itf04g30630), starch and sucrose metabolism (itf03g13270, itf15g09320), carbohydrate metabolism (itf14g10250), carbohydrate and amino acid metabolism (itf12g19270), and amino sugar and nucleotide sugar metabolism (itf03g21950, itf15g04880). This results indicated that sugar and starch are important characteristics to determine the genetic diversity of sweet potatoes. These findings not only illustrate the importance of component traits to genotyping sweet potatoes but also explain an important reason resulting in genetic diversity of sweet potato.

BETA-AMYLASE9 is a plastidial nonenzymatic regulator of leaf starch degradation.

ß-Amylases (BAMs) are key enzymes of transitory starch degradation in chloroplasts, a process that buffers the availability of photosynthetically fixed carbon over the diel cycle to maintain energy levels and plant growth at night. However, during vascular plant evolution, the BAM gene family diversified, giving rise to isoforms with different compartmentation and biological activities. Here, we characterized BETA-AMYLASE 9 (BAM9) of Arabidopsis (Arabidopsis thaliana). Among the BAMs, BAM9 is most closely related to BAM4 but is more widely conserved in plants. BAM9 and BAM4 share features including their plastidial localization and lack of measurable α-1,4-glucan hydrolyzing capacity. BAM4 is a regulator of starch degradation, and bam4 mutants display a starch-excess phenotype. Although bam9 single mutants resemble the wild-type (WT), genetic experiments reveal that the loss of BAM9 markedly enhances the starch-excess phenotypes of mutants already impaired in starch degradation. Thus, BAM9 also regulates starch breakdown, but in a different way. Interestingly, BAM9 gene expression is responsive to several environmental changes, while that of BAM4 is not. Furthermore, overexpression of BAM9 in the WT reduced leaf starch content, but overexpression in bam4 failed to complement fully that mutant's starch-excess phenotype, suggesting that BAM9 and BAM4 are not redundant. We propose that BAM9 activates starch degradation, helping to manage carbohydrate availability in response to fluctuations in environmental conditions. As such, BAM9 represents an interesting gene target to explore in crop species.

OsMADS1 Regulates Grain Quality, Gene Expressions, and Regulatory Networks of Starch and Storage Protein Metabolisms in Rice.

OsMADS1 plays a vital role in regulating floret development and grain shape, but whether it regulates rice grain quality still remains largely unknown. Therefore, we used comprehensive molecular genetics, plant biotechnology, and functional omics approaches, including phenotyping, mapping-by-sequencing, target gene seed-specific RNAi, transgenic experiments, and transcriptomic profiling to answer this biological and molecular question. Here, we report the characterization of the 'Oat-like rice' mutant, with poor grain quality, including chalky endosperms, abnormal morphology and loose arrangement of starch granules, and lower starch content but higher protein content in grains. The poor grain quality of Oat-like rice was found to be caused by the mutated OsMADS1Olr allele through mapping-by-sequencing analysis and transgenic experiments. OsMADS1 protein is highly expressed in florets and developing seeds. Both OsMADS1-eGFP and OsMADS1Olr-eGFP fusion proteins are localized in the nucleus. Moreover, seed-specific RNAi of OsMADS1 also caused decreased grain quality in transgenic lines, such as the Oat-like rice. Further transcriptomic profiling between Oat-like rice and Nipponbare grains revealed that OsMADS1 regulates gene expressions and regulatory networks of starch and storage protein metabolisms in rice grains, hereafter regulating rice quality. In conclusion, our results not only reveal the crucial role and preliminary mechanism of OsMADS1 in regulating rice grain quality but also highlight the application potentials of OsMADS1 and the target gene seed-specific RNAi system in improving rice grain quality by molecular breeding.

Genetic Engineering of Starch Biosynthesis in Maize Seeds for Efficient Enzymatic Digestion of Starch during Bioethanol Production.

Maize accumulates large amounts of starch in seeds which have been used as food for human and animals. Maize starch is an importantly industrial raw material for bioethanol production. One critical step in bioethanol production is degrading starch to oligosaccharides and glucose by α-amylase and glucoamylase. This step usually requires high temperature and additional equipment, leading to an increased production cost. Currently, there remains a lack of specially designed maize cultivars with optimized starch (amylose and amylopectin) compositions for bioethanol production. We discussed the features of starch granules suitable for efficient enzymatic digestion. Thus far, great advances have been made in molecular characterization of the key proteins involved in starch metabolism in maize seeds. The review explores how these proteins affect starch metabolism pathway, especially in controlling the composition, size and features of starch. We highlight the roles of key enzymes in controlling amylose/amylopectin ratio and granules architecture. Based on current technological process of bioethanol production using maize starch, we propose that several key enzymes can be modified in abundance or activities via genetic engineering to synthesize easily degraded starch granules in maize seeds. The review provides a clue for developing special maize cultivars as raw material in the bioethanol industry.

Genome-Wide Investigation of BAM Gene Family in Annona atemoya: Evolution and Expression Network Profiles during Fruit Ripening.

ß-amylase proteins (BAM) are important to many aspects of physiological process such as starch degradation. However, little information was available about the BAM genes in Annona atemoya, an important tropical fruit. Seven BAM genes containing the conservative domain of glycoside hydrolase family 14 (PF01373) were identified with Annona atemoya genome, and these BAM genes can be divided into four groups. Subcellular localization analysis revealed that AaBAM3 and AaBAM9 were located in the chloroplast, and AaBAM1.2 was located in the cell membrane and the chloroplast. The AaBAMs belonging to Subfamily I contribute to starch degradation have the higher expression than those belonging to Subfamily II. The analysis of the expression showed that AaBAM3 may function in the whole fruit ripening process, and AaBAM1.2 may be important to starch degradation in other organs. Temperature and ethylene affect the expression of major AaBAM genes in Subfamily I during fruit ripening. These expressions and subcellular localization results indicating ß-amylase play an important role in starch degradation.

TaNAC100 acts as an integrator of seed protein and starch synthesis exerting pleiotropic effects on agronomic traits in wheat.

High-molecular-weight glutenin subunits (HMW-GS) are major components of seed storage proteins (SSPs) and largely determine the processing properties of wheat (Triticum aestivum) flour. HMW-GS are encoded by the GLU-1 loci and regulated at the transcriptional level by interaction between cis-elements and transcription factors (TFs). We recently validated the function of conserved cis-regulatory modules (CCRMs) in GLU-1 promoters, but their interacting TFs remained uncharacterized. Here we identified a CCRM-binding NAM-ATAF-CUC (NAC) protein, TaNAC100, through yeast one-hybrid (Y1H) library screening. Transactivation assays demonstrated that TaNAC100 could bind to the GLU-1 promoters and repress their transcription activity in tobacco (Nicotiana benthamiana). Overexpression of TaNAC100 in wheat significantly reduced the contents of HMW-GS and other SSPs as well as total seed protein. This was confirmed by transcriptome analyses. Conversely, enhanced expression of TaNAC100 increased seed starch contents and expression of key starch synthesis-related genes, such as TaGBSS1 and TaSUS2. Y1H assays also indicated TaNAC100 binding with the promoters of TaGBSS1 and TaSUS2. These results suggest that TaNAC100 functions as a hub controlling seed protein and starch synthesis. Phenotypic analyses showed that TaNAC100 overexpression repressed plant height, increased heading date, and promoted seed size and thousand kernel weight. We also investigated sequence variations in a panel of cultivars, but did not identify significant association of TaNAC100 haplotypes with agronomic traits. The findings not only uncover a useful gene for wheat breeding but also provide an entry point to reveal the mechanism underlying metabolic balance of seed storage products.

Overexpression of a wheat α-amylase type 2 impact on starch metabolism and abscisic acid sensitivity during grain germination.

Despite being of vital importance for seed establishment and grain quality, starch degradation remains poorly understood in organs such as cereal or legume seeds. In cereals, starch degradation requires the synergetic action of different isoforms of α-amylases. Ubiquitous overexpression of TaAmy2 resulted in a 2.0-437.6-fold increase of total α-amylase activity in developing leaf and harvested grains. These increases led to dramatic alterations of starch visco-properties and augmentation of soluble carbohydrate levels (mainly sucrose and α-gluco-oligosaccharide) in grain. Interestingly, the overexpression of TaAMY2 led to an absence of dormancy in ripened grain due to abscisic acid (ABA) insensitivity. Using an allosteric α-amylase inhibitor (acarbose), we demonstrated that ABA insensitivity was due to the increased soluble carbohydrate generated by the α-amylase excess. Independent from the TaAMY2 overexpression, inhibition of α-amylase during germination led to the accumulation of soluble α-gluco-oligosaccharides without affecting the first stage of germination. These findings support the hypotheses that (i) endosperm sugar may overcome ABA signalling and promote sprouting, and (ii) α-amylase may not be required for the initial stage of grain germination, an observation that questions the function of the amylolytic enzyme in the starch degradation process during germination.

Laser microdissection transcriptome data derived gene regulatory networks of developing rice endosperm revealed tissue- and stage-specific regulators modulating starch metabolism.

KEY MESSAGE: Laser microdissection applied on the developing rice endosperm revealed tissue- and stage-specific regulators modulating programmed cell death and desiccation tolerance mechanisms in the central starchy endosperm following starch metabolism. Rice (Oryza sativa L.) filial seed tissues are heterozygous in its function, which accumulate distinct storage compounds spatially in starchy endosperm and aleurone. In this study, we identified the 18 tissue- and stage-specific gene co-regulons in the developing endosperm by isolating four fine tissues dorsal aleurone layer (AL), central starchy endosperm (CSE), dorsal starchy endosperm (DSE), and lateral starchy endosperm (LSE) at two developmental stages (7 days after flowering, DAF and 12DAF) using laser microdissection (LM) coupled with gene expression analysis of a 44 K microarray. The derived co-expression regulatory networks depict that distinct set of starch biosynthesis genes expressed preferentially at first in CSE at 7 DAF and extend its spatial expression to LSE and DSE by 12 DAF. Interestingly, along with the peak of starch metabolism we noticed accumulation of transcripts related to phospholipid and glycolipid metabolism in CSE during 12 DAF. The spatial distribution of starch accumulation in distinct zones of starchy endosperm contains specific transcriptional factors and hormonal-regulated genes. Genes related to programmed cell death (PCD) were specifically expressed in CSE at 12DAF, when starch accumulation was already completed in that tissue. The aleurone layer present in the outermost endosperm accumulates transcripts of lipid, tricarboxylic acid metabolism, several transporters, while starch metabolism and PCD is not pronounced. These regulatory cascades are likely to play a critical role in determining the positional fate of cells and offer novel insights into the molecular physiological mechanisms of endosperm development from early to middle storage phase.

Identifying loci controlling total starch content of leaf in Nicotiana tabacum through genome-wide association study.

Starch is an important primary metabolite in plants, which can provide bioenergy for fuel ethanol production. There are many studies focusing on starch metabolism in Arabidopsis, maize, and rice, but few reports have been made on the starch content of tobacco leaves. Hence, to identify the marker-trait associations and isolate the candidate genes related to starch content of tobacco leaf, the genome-wide association study (GWAS) was performed using a multiparent advanced generation intercross (MAGIC) population consisting of 276 accessions genotyped by a 430 K SNP array. In this study, we detected the leaf starch content of tobacco plants cultivated in two places (Zhucheng and Chenzhou), which showed a wide variation of starch content in the population. A total of 28 and 45 significant single-nucleotide polymorphism (SNP) loci associated with leaf starch content were identified by single-locus and multi-locus GWAS models, respectively, and the phenotypic variance explained by these loci varied from 1.80 to - 14.73%. Furthermore, among these quantitative trait loci (QTLs), one SNP, AX-106011713 located on chromosome 19, was detected repeatedly in multiple models and two environments, which was selected for linkage disequilibrium (LD) analysis to obtain the target candidate region. Through gene annotation, haplotype, and gene expression analysis, two candidate genes encoding E3 ubiquitin-protein ligase (Ntab0823160) and fructose-bisphosphate aldolase (Ntab0375050) were obtained. Results showed that the variety carrying the beneficial alleles of the two candidate genes had higher gene expression level and leaf starch content, suggesting the potential role of candidate genes in enhancing the level of tobacco leaf starch content. Furthermore, silencing of Ntab0823160 in tobacco leaves reduced the content of total starch to 39.41-69.75% of that in the wide type plants. Taken together, our results provide useful resources for further investigation of the starch metabolic pathway and are also beneficial for the creation of eco-friendly cultivars with increased accumulation of leaf starch content.

Analysis of gene expression in early seed germination of rice: landscape and genetic regulation.

BACKGROUND: Seed germination is a crucial process, which determines the initiation of seed plant life cycle. The early events during this important life cycle transition that called early seed germination is defined as initially water uptake plus radicle growing out of the covering seed layers. However, a specific genome-wide analysis of early seed germination in rice is still obscure. RESULTS: In this study, the physiological characteristics of rice seed during seed germination are determined to define key points of early seed germination. Transcriptome analyses of early phase of seed germination provided deeper insight into the genetic regulation landscape. Many genes involved in starch-to-sucrose transition were differentially expressed, especially alpha-amylase 1b and beta-amylase 2, which were predominantly expressed. Differential exon usage (DEU) genes were identified, which were significantly enriched in the pathway of starch and sucrose metabolism, indicating that DEU events were critical for starch-to-sucrose transition at early seed germination. Transcription factors (TFs) were also dramatic expressed, including the abscisic acid (ABA) responsive gene, OsABI5, and gibberellic acid (GA) responsive genes, GAI. Moreover, GAI transactivated GA responsive gene, GAMYB in vivo, indicating a potential pathway involved in early seed germination process. In addition, CBL-interacting protein kinase (CIPK) genes, such as CIPK13, CIPK14 and CIPK17 were potentially interacted with other proteins, indicating its pivotal role at early seed germination. CONCLUSION: Taken together, gene regulation of early seed germination in rice was complex and protein-to-gene or protein-to-protein interactions were indispensable.

Transcriptional and physiological data revealed cold tolerance in a photo-thermo sensitive genic male sterile line Yu17S.

BACKGROUND: Rice is highly sensitive to chilling stress during the seedling stage. However, the adaptable photo-thermo sensitive genic male sterile (PTGMS) rice line, Yu17S, exhibits tolerance to low temperatures. Currently, the molecular characteristics of Yu17S are unclear. RESULTS: To evaluate the molecular mechanisms behind cold responses in rice seedlings, a comparative transcriptome analysis was performed in Yu17S during seedling development under normal temperature and low temperature conditions. In total, 9317 differentially expressed genes were detected. Gene ontology and pathway analyses revealed that these genes were involved mostly in photosynthesis, carotenoid biosynthesis, carbohydrate metabolism and plant hormone signal transduction. An integrated analysis of specific pathways combined with physiological data indicated that rice seedlings improved the performance of photosystem II when exposed to cold conditions. Genes involved in starch degradation and sucrose metabolism were activated in rice plants exposed to cold stress treatments, which was accompanied by the accumulation of soluble sugar, trehalose, raffinose and galactinol. Furthermore, chilling stress induced the expression of phytoene desaturase, 15-cis-ζ-carotene isomerase, ζ-carotene desaturase, carotenoid isomerase and ß-carotene hydroxylase; this was coupled with the activation of carotenoid synthase activity and increases in abscisic acid (ABA) levels in rice seedlings. CONCLUSIONS: Our results suggest that Yu17S exhibited better tolerance to cold stress with the activation of carotenoid synthase activity and increasing of ABA levels, and as well as the expression of photosynthesis-related genes under cold condition in rice seedlings.

Metabolic flux analysis of the non-transitory starch tradeoff for lipid production in mature tobacco leaves.

The metabolic plasticity of tobacco leaves has been demonstrated via the generation of transgenic plants that can accumulate over 30% dry weight as triacylglycerols. In investigating the changes in carbon partitioning in these high lipid-producing (HLP) leaves, foliar lipids accumulated stepwise over development. Interestingly, non-transient starch was observed to accumulate with plant age in WT but not HLP leaves, with a drop in foliar starch concurrent with an increase in lipid content. The metabolic carbon tradeoff between starch and lipid was studied using 13CO2-labeling experiments and isotopically nonstationary metabolic flux analysis, not previously applied to the mature leaves of a crop. Fatty acid synthesis was investigated through assessment of acyl-acyl carrier proteins using a recently derived quantification method that was extended to accommodate isotopic labeling. Analysis of labeling patterns and flux modeling indicated the continued production of unlabeled starch, sucrose cycling, and a significant contribution of NADP-malic enzyme to plastidic pyruvate production for the production of lipids in HLP leaves, with the latter verified by enzyme activity assays. The results suggest an inherent capacity for a developmentally regulated carbon sink in tobacco leaves and may in part explain the uniquely successful leaf lipid engineering efforts in this crop.

Cytosolic proline is required for basal freezing tolerance in Arabidopsis.

The amino acid proline accumulates in many plant species under abiotic stress conditions, and various protective functions have been proposed. During cold stress, however, proline content in Arabidopsis thaliana does not correlate with freezing tolerance. Freezing sensitivity of a starchless plastidic phosphoglucomutase mutant (pgm) indicated that localization of proline in the cytosol might stabilize the plasma membrane during freeze-thaw events. Here, we show that re-allocation of proline from cytosol to vacuole was similar in the pyrroline-5-carboxylate synthase 2-1 (p5cs2-1) mutant and the pgm mutant and caused similar reduction of basal freezing tolerance. In contrast, the starch excess 1-1 mutant (sex1-1) had even lower freezing tolerance than pgm but did not affect sub-cellular localization of proline. Freezing sensitivity of sex1-1 mutants affected primarily the photosynthetic electron transport and was enhanced in a sex1-1::p5cs2-1 double mutant. These findings indicate that several independent factors determine basal freezing tolerance. In a pgm::p5cs2-1 double mutant, freezing sensitivity and proline allocation to the vacuole were the same as in the parental lines, indicating that the lack of cytosolic proline was the common cause of reduced basal freezing tolerance in both mutants. We conclude that cytosolic proline is an important factor in freezing tolerance of non-acclimated plants.