Ocimum kilimandscharicum 4CL11 negatively regulates adventitious root development via accumulation of flavonoid glycosides.

4-Coumarate-CoA Ligase (4CL) is an important enzyme in the phenylpropanoid biosynthesis pathway. Multiple 4CLs are identified in Ocimum species; however, their in planta functions remain enigmatic. In this study, we independently overexpressed three Ok4CL isoforms from Ocimum kilimandscharicum (Ok4CL7, -11, and -15) in Nicotiana benthamiana. Interestingly, Ok4CL11 overexpression (OE) caused a rootless or reduced root growth phenotype, whereas overexpression of Ok4CL15 produced normal adventitious root (AR) growth. Ok4CL11 overexpression in N. benthamiana resulted in upregulation of genes involved in flavonoid biosynthesis and associated glycosyltransferases accompanied by accumulation of specific flavonoid-glycosides (kaempferol-3-rhamnoside, kaempferol-3,7-O-bis-alpha-l-rhamnoside [K3,7R], and quercetin-3-O-rutinoside) that possibly reduced auxin levels in plants, and such effects were not seen for Ok4CL7 and -15. Docking analysis suggested that auxin transporters (PINs/LAXs) have higher binding affinity to these specific flavonoid-glycosides, and thus could disrupt auxin transport/signaling, which cumulatively resulted in a rootless phenotype. Reduced auxin levels, increased K3,7R in the middle and basal stem sections, and grafting experiments (intra and inter-species) indicated a disruption of auxin transport by K3,7R and its negative effect on AR development. Supplementation of flavonoids and the specific glycosides accumulated by Ok4CL11-OE to the wild-type N. benthamiana explants delayed the AR emergence and also inhibited AR growth. While overexpression of all three Ok4CLs increased lignin accumulation, flavonoids, and their specific glycosides were accumulated only in Ok4CL11-OE lines. In summary, our study reveals unique indirect function of Ok4CL11 to increase specific flavonoids and their glycosides, which are negative regulators of root growth, likely involved in inhibition of auxin transport and signaling.

Tandem Expression of a Mobile RNA and Its RNA-Binding Protein(s) Enhances Tuber Productivity in Potato.

A significant number of discoveries in past two decades have established the importance of long-distance signaling in controlling plant growth, development, and biotic and abiotic stress responses. Numerous mobile signals, such as mRNAs, proteins, including RNA-binding proteins, small RNAs, sugars, and phytohormones, are shown to regulate various agronomic traits such as flowering, fruit, seed development, and tuberization. Potato is a classic model tuber crop, and several mobile signals are known to govern tuber development. However, it is unknown if these mobile signals have any synergistic effects on potato crop improvement. Here, we employed a simple innovative strategy to test the cumulative effects of a key mobile RNA, StBEL5, and its RNA-binding proteins, StPTB1, and -6 on tuber productivity of two potato cultivars, Solanum tuberosum cv. Désirée and subspecies andigena, using a multi-gene stacking approach. In this approach, the coding sequences of StBEL5 and StPTB1/6 are driven by their respective native promoters to efficiently achieve targeted expression in phloem for monitoring tuber productivity. We demonstrate that this strategy resulted in earliness for tuberization and enhanced tuber productivity by 2-4 folds under growth chamber, greenhouse, and field conditions. This multi-gene stacking approach could be adopted to other crops, whose agronomic traits are governed by mobile macromolecules, expanding the possibilities to develop crops with improved traits and enhanced yields.

The phased short-interfering RNA siRD29(-) regulates GIBBERELLIN 3-OXIDASE 3 during stolon-to-tuber transitions in potato.

Phased short-interfering RNAs (phasiRNAs) fine tune various stages of growth, development, and stress responses in plants. Potato (Solanum tuberosum) tuberization is a complex process, wherein a belowground modified stem (stolon) passes through developmental stages like swollen stolon and minituber before it matures to a potato. Previously, we identified several phasiRNA-producing loci (PHAS) from stolon-to-tuber transition stages. However, whether phasiRNAs mediate tuber development remains unknown. Here, we show that a gene encoding NB-ARC DOMAIN-CONTAINING DISEASE RESISTANCE PROTEIN (StRGA4; a PHAS locus) is targeted by Stu-microRNA482c to generate phasiRNAs. Interestingly, we observed that one of the phasiRNAs, referred as short-interfering RNA D29(-), i.e. siRD29(-), targets the gibberellin (GA) biosynthesis gene GIBBERELLIN 3-OXIDASE 3 (StGA3ox3). Since regulation of bioactive GA levels in stolons controls tuber development, we hypothesized that a gene regulatory module, Stu-miR482c-StRGA4-siRD29(-)-StGA3ox3, could govern tuber development. Through transient expression assays and small RNA sequencing, generation of siRD29(-) and its phase was confirmed in planta. Notably, the expression of StGA3ox3 was higher in swollen stolon compared to stolon, whereas siRD29(-) showed a negative association with StGA3ox3 expression. Antisense (AS) lines of StGA3ox3 produced more tubers compared to wild type. As expected, StRGA4 overexpression (OE) lines had high levels of siRD29(-) and mimicked the phenotypes of StGA3ox3-AS lines, indicating the functionality of this module in potato. In vitro tuberization assays (with or without a GA inhibitor) using StGA3ox3 antisense lines and overexpression lines of StGA3ox3 or StRGA4 revealed that StGA3ox3 controls the tuber stalk development. Taken together, our findings suggest that a phasiRNA, siRD29(-), mediates the regulation of StGA3ox3 during stolon-to-tuber transitions in potato.

Development of aerial and belowground tubers in potato is governed by photoperiod and epigenetic mechanism.

Plants exhibit diverse developmental plasticity and modulate growth responses under various environmental conditions. Potato (Solanum tuberosum), a modified stem and an important food crop, serves as a substantial portion of the world's subsistence food supply. In the past two decades, crucial molecular signals have been identified that govern the tuberization (potato development) mechanism. Interestingly, microRNA156 overexpression in potato provided the first evidence for induction of profuse aerial stolons and tubers from axillary meristems under short-day (SD) photoperiod. A similar phenotype was noticed for overexpression of epigenetic modifiers-MUTICOPY SUPRESSOR OF IRA1 (StMSI1) or ENAHNCER OF ZESTE 2 (StE[z]2), and knockdown of B-CELL-SPECIFIC MOLONEY MURINE LEUKEMIA VIRUS INTEGRATION SITE 1 (StBMI1). This striking phenotype represents a classic example of modulation of plant architecture and developmental plasticity. Differentiation of a stolon to a tuber or a shoot under in vitro or in vivo conditions symbolizes another example of organ-level plasticity and dual fate acquisition in potato. Stolon-to-tuber transition is governed by SD photoperiod, mobile RNAs/proteins, phytohormones, a plethora of small RNAs and their targets. Recent studies show that polycomb group proteins control microRNA156, phytohormone metabolism/transport/signaling and key tuberization genes through histone modifications to govern tuber development. Our comparative analysis of differentially expressed genes between the overexpression lines of StMSI1, StBEL5 (BEL1-LIKE transcription factor [TF]), and POTATO HOMEOBOX 15 TF revealed more than 1,000 common genes, indicative of a mutual gene regulatory network potentially involved in the formation of aerial and belowground tubers. In this review, in addition to key tuberization factors, we highlight the role of photoperiod and epigenetic mechanism that regulates the development of aerial and belowground tubers in potato.

Diversity in Chemical Structures and Biological Properties of Plant Alkaloids.

Phytochemicals belonging to the group of alkaloids are signature specialized metabolites endowed with countless biological activities. Plants are armored with these naturally produced nitrogenous compounds to combat numerous challenging environmental stress conditions. Traditional and modern healthcare systems have harnessed the potential of these organic compounds for the treatment of many ailments. Various chemical entities (functional groups) attached to the central moiety are responsible for their diverse range of biological properties. The development of the characterization of these plant metabolites and the enzymes involved in their biosynthesis is of an utmost priority to deliver enhanced advantages in terms of biological properties and productivity. Further, the incorporation of whole/partial metabolic pathways in the heterologous system and/or the overexpression of biosynthetic steps in homologous systems have both become alternative and lucrative methods over chemical synthesis in recent times. Moreover, in-depth research on alkaloid biosynthetic pathways has revealed numerous chemical modifications that occur during alkaloidal conversions. These chemical reactions involve glycosylation, acylation, reduction, oxidation, and methylation steps, and they are usually responsible for conferring the biological activities possessed by alkaloids. In this review, we aim to discuss the alkaloidal group of plant specialized metabolites and their brief classification covering major categories. We also emphasize the diversity in the basic structures of plant alkaloids arising through enzymatically catalyzed structural modifications in certain plant species, as well as their emerging diverse biological activities. The role of alkaloids in plant defense and their mechanisms of action are also briefly discussed. Moreover, the commercial utilization of plant alkaloids in the marketplace displaying various applications has been enumerated.

Auxin: An emerging regulator of tuber and storage root development.

Many tuber and storage root crops owing to their high nutritional values offer high potential to overcome food security issues. The lack of information regarding molecular mechanisms that govern belowground storage organ development (except a tuber crop, potato) has limited the application of biotechnological strategies for improving storage crop yield. Phytohormones like gibberellin and cytokinin are known to play a crucial role in governing potato tuber development. Another phytohormone, auxin has been shown to induce tuber initiation and growth, and its crosstalk with gibberellin and strigolactone in a belowground modified stem (stolon) contributes to the overall potato tuber yield. In this review, we describe the crucial role of auxin biology in development of potato tubers. Considering the emerging reports from commercially important storage root crops (sweet potato, cassava, carrot, sugar beet and radish), we propose the function of auxin and related gene regulatory network in storage root development. The pattern of auxin content of stolon during various stages of potato tuber formation appears to be consistent with its level in various developmental stages of storage roots. We have also put-forward the potential of three-way interaction between auxin, strigolactone and mycorrhizal fungi in tuber and storage root development. Overall, we propose that auxin gene regulatory network and its crosstalk with other phytohormones in stolons/roots could govern belowground tuber and storage root development.

A historical overview of long-distance signalling in plants.

Be it a small herb or a large tree, intra- and intercellular communication and long-distance signalling between distant organs are crucial for every aspect of plant development. The vascular system, comprising xylem and phloem, acts as a major conduit for the transmission of long-distance signals in plants. In addition to expanding our knowledge of vascular development, numerous reports in the past two decades revealed that selective populations of RNAs, proteins, and phytohormones function as mobile signals. Many of these signals were shown to regulate diverse physiological processes, such as flowering, leaf and root development, nutrient acquisition, crop yield, and biotic/abiotic stress responses. In this review, we summarize the significant discoveries made in the past 25 years, with emphasis on key mobile signalling molecules (mRNAs, proteins including RNA-binding proteins, and small RNAs) that have revolutionized our understanding of how plants integrate various intrinsic and external cues in orchestrating growth and development. Additionally, we provide detailed insights on the emerging molecular mechanisms that might control the selective trafficking and delivery of phloem-mobile RNAs to target tissues. We also highlight the cross-kingdom movement of mobile signals during plant-parasite relationships. Considering the dynamic functions of these signals, their implications in crop improvement are also discussed.

The Polycomb group methyltransferase StE(z)2 and deposition of H3K27me3 and H3K4me3 regulate the expression of tuberization genes in potato.

Polycomb repressive complex (PRC) group proteins regulate various developmental processes in plants by repressing target genes via H3K27 trimethylation, and they function antagonistically with H3K4 trimethylation mediated by Trithorax group proteins. Tuberization in potato has been widely studied, but the role of histone modifications in this process is unknown. Recently, we showed that overexpression of StMSI1, a PRC2 member, alters the expression of tuberization genes in potato. As MSI1 lacks histone-modification activity, we hypothesized that this altered expression could be caused by another PRC2 member, StE(z)2, a potential H3K27 methyltransferase in potato. Here, we demonstrate that a short-day photoperiod influences StE(z)2 expression in the leaves and stolons. StE(z)2 overexpression alters plant architecture and reduces tuber yield, whereas its knockdown enhances yield. ChIP-sequencing using stolons induced by short-days indicated that several genes related to tuberization and phytohormones, such as StBEL5/11/29, StSWEET11B, StGA2OX1, and StPIN1 carry H3K4me3 or H3K27me3 marks and/or are StE(z)2 targets. Interestingly, we observed that another important tuberization gene, StSP6A, is targeted by StE(z)2 in leaves and that it has increased deposition of H3K27me3 under long-day (non-induced) conditions compared to short days. Overall, our results show that StE(z)2 and deposition of H3K27me3 and/or H3K4me3 marks might regulate the expression of key tuberization genes in potato.

Molecular signals that govern tuber development in potato.

The potato serves as the fourth most important food crop on the planet after the three cereal crops. It is rich in starch, storage proteins and important vitamins, dietary antioxidants and minerals. Potato is a modified stem (stolon) that grows underground, at the base of the plant, under favourable conditions. Perception and processing of signals occur in leaves and the corresponding information is transported to the stolon-tip. The elongation of the stolon-tip ceases and the plane of cell division changes from transverse to longitudinal, causing swelling of the sub-apical region of the stolon. This is accompanied by synthesis of starch in leaves, followed by its transport to and accumulation in the stolon. The initiation of tuber developmental signals and the subsequent stolon-to-tuber transition (tuberization) is undoubtedly a dynamic process which involves integration of multiple molecular factors, environmental cues and crosstalk between various pathways, including phytohormones. Understanding the tuberization process has been an aim of many plant biologists across the globe. Recent discoveries have shown that apart from photoperiod and hormonal metabolism, there are crucial transcription factors, small RNAs, full-length mobile mRNAs and proteins that regulate tuberization in potato. Although we have gained significant knowledge about the tuberization process, many questions on the underlying mechanisms of tuber development remain to be answered. In this review, we summarize the crucial molecular signals that govern tuber formation and propose an updated tuberization network along with future research directions.

BEL1-like protein (StBEL5) regulates CYCLING DOF FACTOR1 (StCDF1) through tandem TGAC core motifs in potato.

Tuberization in potato is governed by many intrinsic and extrinsic factors. Various molecular signals, such as red light photoreceptor (StPHYB), BEL1-like transcription factor (StBEL5), CYCLING DOF FACTOR1 (StCDF1), StCO1/2 (CONSTANS1/2) and StSP6A (Flowering Locus T orthologue), function as crucial regulators during the photoperiod-dependent tuberization pathway. StCDF1 induces tuberization by increasing StSP6A levels via StCO1/2 suppression. Although the circadian clock proteins, GIGANTEA (StGI) and FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (StFKF1), are reported as StCDF1 interactors, how the StCDF1 gene is regulated in potato is unknown. The BEL-KNOX heterodimer regulates key tuberization genes through tandem TGAC core motifs in their promoters. A recent study reported the presence of six tandem TGAC core motifs in the StCDF1 promoter, suggesting possible regulation of StCDF1 by StBEL5. In our study, we observed a positive correlation between StBEL5 and StCDF1 expression, whereas StCDF1 and its known repressor, StFKF1, showed a negative correlation for the tested tissue types. To investigate the StBEL5-StCDF1 interaction, we generated transgenic potato promoter lines containing a wild-type or mutated (deletion of six tandem TGAC sites) StCDF1 promoter fused to GUS. Wild-type promoter transgenic lines exhibited widespread GUS activity, whereas this activity was absent in the mutated promoter transgenic lines. Moreover, StBEL5 and StCDF1 transcript levels were significantly higher in the stolon-to-tuber stages under short-day conditions compared to long-day conditions. Using wild-type and mutated prStCDF1 as baits in Y1H assays, we further demonstrated that StBEL5 interacts with the StCDF1 promoter through tandem TGAC motifs, indicating direct regulation of StCDF1 by StBEL5 in potato.

Mobile RNAs and proteins: Prospects in storage organ development of tuber and root crops.

Storage tuber and root crops make up a significant portion of the world's subsistence food supply. Because of their importance in food security, yield enhancement has become a priority. A major focus has been to understand the biology of belowground storage organ development. Considerable insights have been gained studying tuber development in potato. We now know that two mobile signals, a full-length mRNA, StBEL5, and a protein, StSP6A, play pivotal roles in regulating tuber development. Under favorable conditions, these signals move from leaves to a belowground modified stem (stolon) and regulate genes that activate tuberization. Overexpression of StBEL5 or StSP6A increases tuber yield even under non-inductive conditions. The mRNAs of two close homologs of StBEL5, StBEL11 and StBEL29, are also known to be mobile but act as repressors of tuberization. Polypyrimidine tract-binding proteins (PTBs) are RNA-binding proteins that facilitate the movement of these mRNAs. Considering their role in tuberization, it is possible that these mobile signals play a major role in storage root development as well. In this review, we explore the presence of these signals and their relevance in the development and yield potential of several important storage root crops.

Conservation of polypyrimidine tract binding proteins and their putative target RNAs in several storage root crops.

BACKGROUND: Polypyrimidine-tract binding proteins (PTBs) are ubiquitous RNA-binding proteins in plants and animals that play diverse role in RNA metabolic processes. PTB proteins bind to target RNAs through motifs rich in cytosine/uracil residues to fine-tune transcript metabolism. Among tuber and root crops, potato has been widely studied to understand the mobile signals that activate tuber development. Potato PTBs, designated as StPTB1 and StPTB6, function in a long-distance transport system by binding to specific mRNAs (StBEL5 and POTH1) to stabilize them and facilitate their movement from leaf to stolon, the site of tuber induction, where they activate tuber and root growth. Storage tubers and root crops are important sustenance food crops grown throughout the world. Despite the availability of genome sequence for sweet potato, cassava, carrot and sugar beet, the molecular mechanism of root-derived storage organ development remains completely unexplored. Considering the pivotal role of PTBs and their target RNAs in potato storage organ development, we propose that a similar mechanism may be prevalent in storage root crops as well. RESULTS: Through a bioinformatics survey utilizing available genome databases, we identify the orthologues of potato PTB proteins and two phloem-mobile RNAs, StBEL5 and POTH1, in five storage root crops - sweet potato, cassava, carrot, radish and sugar beet. Like potato, PTB1/6 type proteins from these storage root crops contain four conserved RNA Recognition Motifs (characteristic of RNA-binding PTBs) in their protein sequences. Further, 3´ UTR (untranslated region) analysis of BEL5 and POTH1 orthologues revealed the presence of several cytosine/uracil motifs, similar to those present in potato StBEL5 and POTH1 RNAs. Using RT-qPCR assays, we verified the presence of these related transcripts in leaf and root tissues of these five storage root crops. Similar to potato, BEL5-, PTB1/6- and POTH1-like orthologue RNAs from the aforementioned storage root crops exhibited differential accumulation patterns in leaf and storage root tissues. CONCLUSIONS: Our results suggest that the PTB1/6-like orthologues and their putative targets, BEL5- and POTH1-like mRNAs, from storage root crops could interact physically, similar to that in potato, and potentially, could function as key molecular signals controlling storage organ development in root crops.

The mobile RNAs, StBEL11 and StBEL29, suppress growth of tubers in potato.

KEY MESSAGE: We demonstrate that RNAs of StBEL11 and StBEL29 are phloem-mobile and function antagonistically to the growth-promoting characteristics of StBEL5 in potato. Both these RNAs appear to inhibit tuber growth by repressing the activity of target genes of StBEL5 in potato. Moreover, upstream sequence driving GUS expression in transgenic potato lines demonstrated that both StBEL11 and -29 promoter activity is robust in leaf veins, petioles, stems, and vascular tissues and induced by short days in leaves and stolons. Steady-state levels of their mRNAs were also enhanced by short-day conditions in selective organs. There are thirteen functional BEL1-like genes in potato that encode for a family of transcription factors (TF) ubiquitous in the plant kingdom. These BEL1 TFs work in tandem with KNOTTED1-types to regulate the expression of numerous target genes involved in hormone metabolism and growth processes. One of the StBELs, StBEL5, functions as a long-distance mRNA signal that is transcribed in leaves and moves into roots and stolons to stimulate growth. The two most closely related StBELs to StBEL5 are StBEL11 and -29. Together these three genes make up more than 70% of all StBEL transcripts present throughout the potato plant. They share a number of common features, suggesting they may be co-functional in tuber development. Upstream sequence driving GUS expression in transgenic potato lines demonstrated that both StBEL11 and -29 promoter activity is robust in leaf veins, petioles, stems, and vascular tissues and induced by short-days in leaves and stolons. Steady-state levels of their mRNAs were also enhanced by short-day conditions in specific organs. Using a transgenic approach and heterografting experiments, we show that both these StBELs inhibit growth in correlation with the long distance transport of their mRNAs from leaves to roots and stolons, whereas suppression lines of these two RNAs exhibited enhanced tuber yields. In summary, our results indicate that the RNAs of StBEL11 and StBEL29 are phloem-mobile and function antagonistically to the growth-promoting characteristics of StBEL5. Both these RNAs appear to inhibit growth in tubers by repressing the activity of target genes of StBEL5.

Regulation, overexpression, and target gene identification of Potato Homeobox 15 (POTH15) - a class-I KNOX gene in potato.

Potato Homeobox 15 (POTH15) is a KNOX-I (Knotted1-like homeobox) family gene in potato that is orthologous to Shoot Meristemless (STM) in Arabidopsis. Despite numerous reports on KNOX genes from different species, studies in potato are limited. Here, we describe photoperiodic regulation of POTH15, its overexpression phenotype, and identification of its potential targets in potato (Solanum tuberosum ssp. andigena). qRT-PCR analysis showed a higher abundance of POTH15 mRNA in shoot tips and stolons under tuber-inducing short-day conditions. POTH15 promoter activity was detected in apical and axillary meristems, stolon tips, tuber eyes, and meristems of tuber sprouts, indicating its role in meristem maintenance and leaf development. POTH15 overexpression altered multiple morphological traits including leaf and stem development, leaflet number, and number of nodes and branches. In particular, the rachis of the leaf was completely reduced and leaves appeared as a bouquet of leaflets. Comparative transcriptomic analysis of 35S::GUS and two POTH15 overexpression lines identified more than 6000 differentially expressed genes, including 2014 common genes between the two overexpression lines. Functional analysis of these genes revealed their involvement in responses to hormones, biotic/abiotic stresses, transcription regulation, and signal transduction. qRT-PCR of selected candidate target genes validated their differential expression in both overexpression lines. Out of 200 randomly chosen POTH15 targets, 173 were found to have at least one tandem TGAC core motif, characteristic of KNOX interaction, within 3.0kb in the upstream sequence of the transcription start site. Overall, this study provides insights to the role of POTH15 in controlling diverse developmental processes in potato.

Quantifying the impact of exogenous abscisic acid and gibberellins on pre-maturity α-amylase formation in developing wheat grains.

To study the role of abscisic acid (ABA) and gibberellins (GA) in pre-maturity α-amylase (PMA) formation in developing wheat grain, two glasshouse experiments were conducted under controlled conditions in the highly PMA-susceptible genotype Rialto. The first, determined the relative efficacy of applying hormone solutions by injection into the peduncle compared to direct application to the intact grain. The second, examined the effects of each hormone, applied by either method, at mid-grain development on PMA in mature grains. In the first experiment, tritiated ABA ((3)H-ABA) and gibberellic acid ((3)H-GA3) were diluted with unlabelled ABA (100 µM) and GA3 (50 µM), respectively, and applied at mid-grain development using both methods. Spikes were harvested after 24, 48 and 72 h from application, and hormone taken up by grains was determined. After 72 h, the uptake per grain in terms of hormones applied was approximately 13% for ABA and 8% for GA3 when applied onto the grains, and approximately 17% for ABA and 5% for GA3 when applied by injection. In the second experiment, applied ABA reduced, whereas applied GA3 increased α-amylase activity. This confirmed that exogenously applied ABA and GA were absorbed in sufficient amounts to alter grain metabolism and impact on PMA.