Biosynthesis of DDMP saponins in soybean is regulated by a distinct UDP-glycosyltransferase.

2,3-Dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) saponins are one of the major saponin groups that are widely distributed in legumes such as pea, barrel medic, chickpea, and soybean. The steps involved in DDMP saponin biosynthesis remain uncharacterized at the molecular level. We isolated two recessive mutants that lack DDMP saponins from an ethyl methanesulfonate-induced mutant population of soybean cultivar Pungsannamul. Segregation analysis showed that the production of DDMP saponins is controlled by a single locus, named Sg-9. The locus was physically mapped to a 130-kb region on chromosome 16. Nucleotide sequence analysis of candidate genes in the region revealed that each mutant has a single-nucleotide polymorphism in the Glyma.16G033700 encoding a UDP-glycosyltransferase UGT73B4. Enzyme assays and mass spectrum-coupled chromatographic analysis reveal that the Sg-9 protein has glycosyltransferase activity, converting sapogenins and group B saponins to glycosylated products, and that mutant proteins had only partial activities. The tissue-specific expression profile of Sg-9 matches the accumulation pattern of DDMP saponins. This is the first report on a new gene and its function in the biosynthesis of DDMP saponins. Our findings indicate that Sg-9 encodes a putative DDMP transferase that plays a critical role in the biosynthesis of DDMP saponins.

GROWTH-REGULATING FACTOR and GRF-INTERACTING FACTOR Specify Meristematic Cells of Gynoecia and Anthers.

We investigated the biological roles of the Arabidopsis (Arabidopsis thaliana) GROWTH-REGULATING FACTOR (GRF) and GRF-INTERACTING FACTOR (GIF) transcriptional complex in the development of gynoecia and anthers. There are nine GRFs and three GIFs in Arabidopsis, and seven GRFs are posttranscriptionally silenced by microRNA396 (miR396). We found that overexpression of MIR396 in the gif1 gif2 double mutant background (gif1 gif2 35S:MIR396) resulted in neither ovary nor pollen. Histological and molecular marker-based analyses revealed that the mutant gynoecial primordia failed to develop carpel margin meristems and mature flowers lacked the ovary, consisting only of the stigma, style, and replum-like tissues. The mutant anther primordia were not able to form the pluripotent archesporial cells that produce pollen mother cells and microsporangia. Multiple combinations of GRF mutations also displayed the same phenotypes, indicating that the GRF-GIF duo is required for the formation of those meristematic and pluripotent cells. Most GRF proteins are localized and abundant in those cells. We also found that the weak gynoecial defects of pinoid-3 (pid-3) mutants were remarkably exacerbated by gif1 gif2 double mutations and 35S:MIR396, so that none of the gynoecia produced by gif1 gif2 pid-3 and 35S:MIR396 pid-3 developed ovaries at all. Moreover, gif1 gif2 double mutations and 35S:MIR396 also acted synergistically with 1-N-naphthylphthalamic acid in forming aberrant gynoecia. The results altogether suggest that the GRF-GIF duo regulates the meristematic and pluripotent competence of carpel margin meristems and the archesporial cell lineage and that this regulation is implemented in association with auxin action, ultimately conferring reproductive competence on Arabidopsis.

THO2, a core member of the THO/TREX complex, is required for microRNA production in Arabidopsis.

The THO/TREX complex mediates transport of nascent mRNAs from the nucleus towards the cytoplasm in animals, and has a role in small interfering RNA-dependent processes in plants. Here we describe five mutant alleles of Arabidopsis thaliana THO2, which encodes a core subunit of the plant THO/TREX complex. tho2 mutants present strong developmental defects resembling those in plants compromised in microRNA (miRNA) activity. In agreement, not only were the levels of siRNAs reduced in tho2 mutants, but also those of mature miRNAs. As a consequence, a feedback mechanism is triggered, increasing the amount of miRNA precursors, and finally causing accumulation of miRNA-targeted mRNAs. Yeast two-hybrid experiments and confocal microscopy showed that THO2 does not appear to interact with any of the known miRNA biogenesis components, but rather with the splicing machinery, implying an indirect role of THO2 in small RNA biogenesis. Using an RNA immunoprecipitation approach, we found that THO2 interacts with miRNA precursors, and that tho2 mutants fail to recruit such precursors into the miRNA-processing complex, explaining the reduction in miRNA production in this mutant background. We also detected alterations in the splicing pattern of genes encoding serine/arginine-rich proteins in tho2 mutants, supporting a previously unappreciated role of the THO/TREX complex in alternative splicing.

The Arabidopsis thaliana GRF-INTERACTING FACTOR gene family plays an essential role in control of male and female reproductive development.

Reproductive success of angiosperms relies on the precise development of the gynoecium and the anther, because their primary function is to bear and to nurture the embryo sac/female gametophyte and pollen, in which the egg and sperm cells, respectively, are generated. It has been known that the GRF-INTERACTING FACTOR (GIF) transcription co-activator family of Arabidopsis thaliana (Arabidopsis) consists of three members and acts as a positive regulator of cell proliferation. Here, we demonstrate that GIF proteins also play an essential role in development of reproductive organs and generation of the gamete cells. The gif1 gif2 gif3 triple mutant, but not the single or double mutants, failed to establish normal carpel margin meristem (CMM) and its derivative tissues, such as the ovule and the septum, resulting in a split gynoecium and no observable embryo sac. The gif triple mutant also displayed severe structural and functional defects in the anther, producing neither microsporangium nor pollen grains. Therefore, we propose that the GIF family of Arabidopsis is a novel and essential component required for the cell specification maintenance during reproductive organ development and, ultimately, for the reproductive competence.

The Arabidopsis thaliana NGATHA transcription factors negatively regulate cell proliferation of lateral organs.

The cell proliferation process of aerial lateral organs, such as leaves and flowers, is coordinated by complex genetic networks that, in general, converge on the cell cycle. The Arabidopsis thaliana NGATHA (AtNGA) family comprises four members that belong to the B3-type transcription factor superfamily, and has been suggested to be involved in growth and development of aerial lateral organs, although its role in the cell proliferation and expansion processes remains to be resolved in more detail. In order to clarify the role of AtNGAs in lateral organ growth, we took a systematic approach using both the loss- and gain-of-functional mutants of all four members. Our results showed that overexpressors of AtNGA1 to AtNGA4 developed small, narrow lateral organs, whereas the nga1 nga2 nga3 nga4 quadruple mutant produced large, wide lateral organs. We found that cell numbers of the lateral organs were significantly affected: a decrease in overexpressors and, inversely, an increase in the quadruple mutant. Kinematic analyses on leaf growth revealed that, compared with the wild type, the overexpressors displayed a lower activity of cell proliferation and yet the mutant a higher activity. Changes in expression of cell cycle-regulating genes were well in accordance with the cell proliferation activities, establishing that the AtNGA transcription factors act as bona fide negative regulators of the cell proliferation of aerial lateral organs.

Regulation of plant growth and development by the GROWTH-REGULATING FACTOR and GRF-INTERACTING FACTOR duo.

Transcription factors are key regulators of gene expression and play pivotal roles in all aspects of living organisms. Therefore, identification and functional characterization of transcription factors is a prerequisite step toward understanding life. This article reviews molecular and biological functions of the two transcription regulator families, GROWTH-REGULATING FACTOR (GRF) and GRF-INTERACTING FACTOR (GIF), which have only recently been recognized. A myriad of experimental evidence clearly illustrates that GRF and GIF are bona fide partner proteins and form a plant-specific transcriptional complex. One of the most conspicuous outcomes from this research field is that the GRF-GIF duo endows the primordial cells of vegetative and reproductive organs with a meristematic specification state, guaranteeing the supply of cells for organogenesis and successful reproduction. It has recently been shown that GIF1 proteins, also known as ANGUSTIFOLIA3, recruit chromatin remodelling complexes to target genes, and that AtGRF expression is directly activated by the floral identity factors, APETALA1 and SEPALLATA3, providing an important insight into understanding of the action of GRF-GIF. Moreover, GRF genes are extensively subjected to post-transcriptional control by microRNA396, revealing the presence of a complex regulatory circuit in regulation of plant growth and development by the GRF-GIF duo.

Abscisic acid prevents the coalescence of protein storage vacuoles by upregulating expression of a tonoplast intrinsic protein gene in barley aleurone.

Tonoplast intrinsic proteins (TIPs) are integral membrane proteins that are known to function in plants as aquaporins. Here, we propose another role for TIPs during the fusion of protein storage vacuoles (PSVs) in aleurone cells, a process that is promoted by gibberellic acid (GA) and prevented by abscisic acid (ABA). Studies of the expression of barley (Hordeum vulgare) TIP genes (HvTIP) showed that GA specifically decreased the abundance of HvTIP1;2 and HvTIP3;1 transcripts, while ABA strongly increased expression of HvTIP3;1. Increased or decreased expression of HvTIP3;1 interfered with the hormonal effects on vacuolation in aleurone protoplasts. HvTIP3;1 gain-of-function experiments delayed GA-induced vacuolation, whereas HvTIP3;1 loss-of-function experiments promoted vacuolation in ABA-treated aleurone cells. These results indicate that TIP plays a key role in preventing the coalescence of small PSVs in aleurone cells. Hormonal regulation of the HvTIP3;1 promoter is similar to the regulation of the endogenous gene, indicating that induction of the transcription of HvTIP3;1 by ABA is a critical factor in the prevention of PSV coalescence in response to ABA. Promoter analysis using deletions and site-directed mutagenesis of sequences identified three cis-acting elements that are responsible for ABA responsiveness in the HvTIP3;1 promoter. Promoter analysis also showed that ABA responsiveness of the HvTIP3;1 promoter is likely to occur via a unique regulatory system distinct from that involving the ABA-response promoter complexes.

A P3A-Type ATPase and an R2R3-MYB Transcription Factor Are Involved in Vacuolar Acidification and Flower Coloration in Soybean.

The determination of flower color mainly depends on the anthocyanin biosynthesis pathway and vacuolar pH; however, unlike the former, the mechanism of vacuolar acidification in soybean remains uncharacterized at the molecular level. To investigate this mechanism, we isolated four recessive purple-blue EMS-induced flower mutants from the purple flower soybean cultivar, Pungsannamul. The petals of all the mutants had increased pH compared with those of wild Pungsannamul. One of the mutants had a single nucleotide substitution in GmPH4, a regulator gene encoding an MYB transcription factor, and the substitution resulted in a premature stop codon in its first exon. The other three mutants had nucleotide substitutions in GmPH5, a single new gene that we identified by physical mapping. It corresponds to Glyma.03G262600 in chromosome 3 and encodes a proton pump that belongs to the P3A-ATPase family. The substitutions resulted in a premature stop codon, which may be a defect in the ATP-binding capacity of GmPH5 and possibly a catalytic inefficiency of GmPH5. The result is consistent with their genetic recessiveness as well as the high pH of mutant petals, suggesting that GmPH5 is directly involved in vacuolar acidification. We also found that the expression of GmPH5 and several putative "acidifying" genes in the gmph4 mutant was remarkably reduced, indicating that GmPH4 may regulate the genes involved in determining the vacuolar pH of soybean petals.

Overexpression of Populus transcription factor PtrTALE12 increases axillary shoot development by regulating WUSCHEL expression.

The TALE (Three Amino acid Loop Extension) transcription factor family has been shown to control meristem formation and organogenesis in plants. To understand the functional roles of the TALE family in woody perennials, each of the TALE members of Populus trichocarpa was overexpressed in Arabidopsis as a proxy. Among them, the overexpression of PtrTALE12 (i.e., 35S::PtrTALE12) resulted in a dramatic increase of axillary shoot development with early flowering. Interestingly, expression of WUSCHEL (WUS), a central regulator of both apical and axillary meristem formation, was significantly increased in the 35S::PtrTALE12 Arabidopsis plants. Conversely, WUS expression was downregulated in 35S::PtrTALE12-SRDX (short transcriptional repressor domain) plants. Further analysis found that PtrTALE12, expressed preferentially in meristem tissues, directly regulates WUS expression in transient activation assays using Arabidopsis leaf protoplast. Yeast two-hybrid assays showed that PtrTALE12 interacts with SHOOT MERISTEMLESS (STM); however, the interaction does not affect the WUS expression. In addition, expression of both CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) genes was suppressed accordingly for early flowering 35S::PtrTALE12 Arabidopsis. Indeed, transgenic poplars overexpressing PtrTALE12 as well as Arabidopsis plants overexpressing AtBLH11, a close homolog of PtrTALE12, phenocopied the 35S::PtrTALE12 Arabidopsis (i.e., increased axillary shoot development). Taken together, our results suggest that PtrTALE12 functions as a positive regulator of axillary shoot formation in both Arabidopsis and poplar.

Overexpression of a Brassica rapa NGATHA gene in Arabidopsis thaliana negatively affects cell proliferation during lateral organ and root growth.

In an effort to elucidate biological functions of transcription factors of Brassica rapa L. (ssp. pekinensis), an NGATHA homolog, BrNGA1, that belongs to the B3-type transcription factor superfamily was identified and expressed in Arabidopsis thaliana under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Arabidopsis plants overexpressing BrNGA1, named BrNGA1ox, displayed markedly reduced organ growth compared with the wild type: lateral organs, such as leaves, flowers and cotyledons, were small and distinctively narrow, and their root growth was also severely retarded. Reduced sizes of BrNGA1ox organs were mainly due to reduction in cell numbers. Kinematic analysis of leaf growth revealed that both the rate and duration of cell proliferation declined during organogenesis, which was consistent with the reduced expression of cyclin genes. Reduction in organ growth was strongly correlated with the small size of meristematic cell pools in the shoot and root meristems. Taken together, these data indicate that BrNGA1 acts as a negative regulator of cell proliferation and may do so, in part, by regulating the size of the meristematic cell pool.

Biological roles and an evolutionary sketch of the GRF-GIF transcriptional complex in plants.

GROWTH-REGULATING FACTORs (GRFs) are sequencepecific DNA-binding transcription factors that regulate various aspects of plant growth and development. GRF proteins interact with a transcription cofactor, GRF-INTERACTING FACTOR (GIF), to form a functional transcriptional complex. For its activities, the GRF-GIF duo requires the SWITCH2/ SUCROSE NONFERMENTING2 chromatin remodeling complex. One of the most conspicuous roles of the duo is conferring the meristematic potential on the proliferative and formative cells during organogenesis. GRF expression is post-transcriptionally down-regulated by microRNA396 (miR396), thus constructing the GRF-GIF-miR396 module and fine-tuning the duo's action. Since the last comprehensive review articles were published over three years ago, many studies have added further insight into its action and elucidated new biological roles. The current review highlights recent advances in our understanding of how the GRF-GIF-miR396 module regulates plant growth and development. In addition, I revise the previous view on the evolutionary origin of the GRF gene family. [BMB Reports 2019; 52(4): 227-238].

Overexpression of AtSGT1, an Arabidopsis salicylic acid glucosyltransferase, leads to increased susceptibility to Pseudomonas syringae.

We reported previously that a recombinant salicylic acid (SA) glucosyltransferase1 (AtSGT1) from Arabidopsis thaliana catalyzes the formation of both SA 2-O-beta-D-glucoside (SAG) and the glucose ester of SA (SGE). Here, transgenic Arabidopsis plants overexpressing AtSGT1 have been constructed, and their phenotypes analyzed. Compared to wild-type plants, transgenic plants showed an increased susceptibility to Pseudomonas syringae and reduced the accumulation levels of both free SA and its glucosylated forms (SAG and SGE). On the other hand, the overexpression increased the levels of methyl salicylate (MeSA) and methyl salicylate 2-O-beta-D-glucoside (MeSAG), and also induced SA carboxyl methyltransferase1 (AtBSMT1) expression, whose products catalyze the conversion of SA to MeSA. Our data indicate that reduced resistance by AtSGT1 overexpression results from a reduction in SA content, which is at least in part caused by increases in MeSAG and MeSA levels at the expense of SA. Our study also suggests that genetic manipulation of AtSGT1 can be utilized as an important regulatory tool for pathogen control.

Molecular elucidation of a new allelic variation at the Sg-5 gene associated with the absence of group A saponins in wild soybean.

In soybean, triterpenoid saponin is one of the major secondary metabolites and is further classified into group A and DDMP saponins. Although they have known health benefits for humans and animals, acetylation of group A saponins causes bitterness and gives an astringent taste to soy products. Therefore, several studies are being conducted to eliminate acetylated group A saponins. Previous studies have isolated and characterized the Sg-5 (Glyma.15g243300) gene, which encodes the cytochrome P450 72A69 enzyme and is responsible for soyasapogenol A biosynthesis. In this study, we elucidated the molecular identity of a novel mutant of Glycine soja, 'CWS5095'. Phenotypic analysis using TLC and LC-PDA/MS/MS showed that the mutant 'CWS5095' did not produce any group A saponins. Segregation analysis showed that the absence of group A saponins is controlled by a single recessive allele. The locus was mapped on chromosome 15 (4.3 Mb) between Affx-89193969 and Affx-89134397 where the previously identified Glyma.15g243300 gene is positioned. Sequence analysis of the coding region for the Glyma.15g243300 gene revealed the presence of four SNPs in 'CWS5095' compared to the control lines. One of these four SNPs (G1127A) leads to the amino acid change Arg376Lys in the EXXR motif, which is invariably conserved among the CYP450 superfamily proteins. Co-segregation analysis showed that the missense mutation (Arg376Lys) was tightly linked with the absence of group A saponins in 'CWS5095'. Even though Arg and Lys have similar chemical features, the 3D modelled protein structure indicates that the replacement of Arg with Lys may cause a loss-of-function of the Sg-5 protein by inhibiting the stable binding of a heme cofactor to the CYP72A69 apoenzyme.

Identification and Molecular Analysis of Four New Alleles at the W1 Locus Associated with Flower Color in Soybean.

In soybean, flavonoid 3'5'-hydroxylase (F3'5'H) and dihydroflavonol-4-reductase (DFR) play a crucial role in the production of anthocyanin pigments. Loss-of-function of the W1 locus, which encodes the former, or W3 and W4, which encode the latter, always produces white flowers. In this study, we searched for new genetic components responsible for the production of white flowers in soybean and isolated four white-flowered mutant lines, i.e., two Glycine soja accessions (CW12700 and CW13381) and two EMS-induced mutants of Glycine max (PE1837 and PE636). F3'5'H expression in CW12700, PE1837, and PE636 was normal, whereas that in CW13381 was aberrant and missing the third exon. Sequence analysis of F3'5'H of CW13381 revealed the presence of an indel (~90-bp AT-repeat) in the second intron. In addition, the F3'5'H of CW12700, PE1837, and PE636 harbored unique single-nucleotide substitutions. The single nucleotide polymorphisms resulted in substitutions of amino acid residues located in or near the SRS4 domain of F3'5'H, which is essential for substrate recognition. 3D structure modeling of F3'5'H indicated that the substitutions could interfere with an interaction between the substrate and heme group and compromise the conformation of the active site of F3'5'H. Recombination analysis revealed a tight correlation between all of the mutant alleles at the W1 locus and white flower color. On the basis of the characterization of the new mutant alleles, we discussed the biological implications of F3'5'H and DFR in the determination of flower colors in soybean.

Conservation and Diversification of the SHR-SCR-SCL23 Regulatory Network in the Development of the Functional Endodermis in Arabidopsis Shoots.

Development of the functional endodermis of Arabidopsis thaliana roots is controlled, in part, by GRAS transcription factors, namely SHORT-ROOT (SHR), SCARECROW (SCR), and SCARECROW-LIKE 23 (SCL23). Recently, it has been shown that the SHR-SCR-SCL23 regulatory module is also essential for specification of the endodermis (known as the bundle sheath) in leaves. Nevertheless, compared with what is known about the role of the SHR-SCR-SCL23 regulatory network in roots, the molecular interactions of SHR, SCR, and SCL23 are much less understood in shoots. Here, we show that SHR forms protein complexes with SCL23 to regulate transcription of SCL23 in shoots, similar to the regulation mode of SCR expression. Our results indicate that SHR acts as master regulator to directly activate the expression of SCR and SCL23. In the SHR-SCR-SCL23 network, we found a previously uncharacterized negative feedback loop whereby SCL23 modulates SHR levels. Through molecular, genetic, physiological, and morphological analyses, we also reveal that the SHR-SCR-SCL23 module plays a key role in the formation of the endodermis (known as the starch sheath) in hypocotyls. Taken together, our results provide new insights into the regulatory role of the SHR-SCR-SCL23 network in the endodermis development in both roots and shoots.

Genetic interaction between GROWTH-REGULATING FACTOR and CUP-SHAPED COTYLEDON in organ separation.

The Arabidopsis thaliana GROWTH-REGULATING FACTOR (GRF) gene family comprises 9 members and encodes a class of transcription factors. We previously demonstrated that GRF genes played an essential role in formation of the boundary region between cotyledons, since their loss-of-function mutants developed fused cotyledons. Our present study shows that the grf mutants display fused floral organs as well. Such fusion phenotypes of embryonic and post-embryonic floral organs are highly reminiscent of the cup-shaped cotyledon (cuc) mutants. In order to test a genetic interaction between GRFs and CUCs, we constructed cuc1 grf1/2/3, cuc2 grf1/2/3, and cuc3 grf1/2/3 quadruple mutants, and found that the mutants showed dramatic increases in cotyledon fusion as well as floral organ fusion. The results suggest that the signaling pathway of GRFs may be genetically associated with that of CUCs in the organ separation process.

Elucidation of Molecular Identity of the W3 Locus and Its Implication in Determination of Flower Colors in Soybean.

The wide range of flower colors in soybean is controlled by six independent loci (W1, W2, W3, W4, Wm, and Wp). Among these loci, mutations in the W3 locus under the w4 allelic background (i.e., w3w4) produce near-white flowers, while the W3w4 genotype produces purple throat flowers. Although a gene encoding dihydroflavonol 4-reductase, DFR1, has been known to be closely associated with the W3 locus, its molecular identity has not yet been characterized. In the present study, we aimed to determine whether DFR1 is responsible for allelic variations in the W3 locus. On the basis of the sequence of a DFR probe, Glyma.14G072700 was identified as a candidate gene for DFR1, and nucleotide sequences of Glyma.14G072700 from cultivars with previously validated genotypes for the W3 locus were determined. As a result, a number of nucleotide polymorphisms, mainly single-base substitutions, between both coding and 5'-upstream region sequences of the W3 and w3 alleles were identified. Among them, an indel of 311-bp in the 5'-upstream region was noteworthy, since the Glyma.14G072700 in all the w3 alleles examined contained the indel, whereas that in all the W3 alleles did not; the former was barely expressed, but the latter was well expressed. These results suggest that Glyma.14G072700 is likely to correspond to DFR1 for the W3 locus and that its expression patterns may lead to allelic color phenotypes of W3 and w3 alleles under the w4 allelic background.

Spatio-temporal distribution patterns of GRF-INTERACTING FACTOR expression and leaf size control.

Developmental biologists have been fascinated with the long-standing mystery of how multicellular organisms, such as plants and animals, sense and control their organ size. In plants, leaves are a suitable experimental system for elucidation of the mystery, because they, like animal organs, inherently exhibit a determinate growth pattern, meaning that they possess genetic information for the control of their final size. The cell proliferation and expansion processes are prerequisites for growth, so that the genetic controls should converge on the 2 cellular processes and decide their rate or duration during leaf growth. Plant scientists have found dozens of genes involved in the control of the cellular processes, including the Arabidopsis thaliana GRF-INTERACTING FACTOR (GIF) family. The GIF family consists of 3 members, GIF1 to GIF3, and encodes a class of transcription co-activators. Although the GIF family genes have been shown to play an essential role in the control of cell proliferation of the leaf organ, understanding of the spatio-temporal behaviors of GIF expression, in both aspects of their promoters and proteins, has been limited to GIF1 (also known as ANGUSTIFOLIA3, AN3). Here, we define kinematic growth properties of wild-type and gif leaf organs and present spatio-temporal expression patterns of all GIF genes, thus providing comprehensive insights into biological roles and expression behaviors of the whole GIF family members during leaf growth.

Evaluation of rice promoters conferring pollen-specific expression in a heterologous system, Arabidopsis.

Promoters can direct gene expression specifically to targeted tissues or cells. Effective with both crop species and model plant systems, these tools can help researchers overcome the practical obstacles associated with transgenic protocols. Here, we identified promoters that allow one to target the manipulation of gene expression during pollen development. Utilizing published transcriptomic databases for rice, we investigated the promoter activity of selected genes in Arabidopsis. From various microarray datasets, including those for anthers and pollen grains at different developmental stages, we selected nine candidate genes that showed high levels of expression in the late stages of rice pollen development. We named these Oryza sativa late pollen-specific genes. Their promoter regions contained various cis-acting elements that could be responsible for anther-/pollen-specific expression. Promoter::GUS-GFP reporters were constructed and introduced into Arabidopsis plants. Histochemical GUS staining revealed that six of the nine rice promoters conferred strong GUS expression that was restricted to the anthers in Arabidopsis. Further analysis showed that although the GUS signals were not detected at the unicellular stage, they strengthened in the bicellular or tricellular stages, peaking at the mature pollen stage. This paralleled their transcriptomic profiles in rice. Based on our results, we proposed that these six rice promoters, which are active in the late stages of pollen formation in the dicot Arabidopsis, can aid molecular breeders in generating new varieties of a monocot plant, rice.

Differential anoxic expression of sugar-regulated genes reveals diverse interactions between sugar and anaerobic signaling systems in rice.

The interaction between the dual roles of sugar as a metabolic fuel and a regulatory molecule was unveiled by examining the changes in sugar signaling upon oxygen deprivation, which causes the drastic alteration in the cellular energy status. In our study, the expression of anaerobically induced genes is commonly responsive to sugar, either under the control of hexokinase or non-hexokinase mediated signaling cascades. Only sugar regulation via the hexokinase pathway was susceptible for O2 deficiency or energy deficit conditions evoked by uncoupler. Examination of sugar regulation of those genes under anaerobic conditions revealed the presence of multiple paths underlying anaerobic induction of gene expression in rice, subgrouped into three distinct types. The first of these, which was found in type-1 genes, involved neither sugar regulation nor additional anaerobic induction under anoxia, indicating that anoxic induction is a simple result from the release of sugar repression by O2-deficient conditions. In contrast, type-2 genes also showed no sugar regulation, albeit with enhanced expression under anoxia. Lastly, expression of type-3 genes is highly enhanced with sugar regulation sustained under anoxia. Intriguingly, the inhibition of the mitochondrial ATP synthesis can reproduce expression pattern of a specific set of anaerobically induced genes, implying that rice cells may sense O2 deprivation, partly via perception of the perturbed cellular energy status. Our study of interaction between sugar signaling and anaerobic conditions has revealed that sugar signaling and the cellular energy status are likely to communicate with each other and influence anaerobic induction of gene expression in rice.