MIR159 regulates multiple aspects of stamen and carpel development and requires dissection and delimitation of differential downstream regulatory network for manipulating fertility traits.

Unravelling genetic networks regulating developmental programs are key to devising and implementing genomics assisted trait modification strategies. It is crucial to understand the role of small RNAs, and the basis of their ability to modify traits. MIR159 has been previously reported to cause defects in anther development in Arabidopsis; however, the complete spectrum and basis of the defects remained unclear. The present study was therefore undertaken to comprehensively investigate the role of miR159 from Brassica juncea in modulating vegetative and reproductive traits. Owing to the polyploid nature of Brassica, paralogous and homeologous copies of MIR159A, MIR159B, and, MIR159C were identified and analysis of the precursor uncovered extensive structural and sequence variation. The MIR159 locus with mature miR159 with perfect target complimentarily with MYB65, was cloned from Brassica juncea var. Varuna for functional characterization by generating constitutively over-expressing lines in Arabidopsis thaliana Col-0. Apart from statistically significant difference in multiple vegetative traits, drastic differences were observed in stamen and pistil. Over-expression of miR159a led to shortening of filament length and loss of tetradynamous condition. Anthers were apiculate, with improper lobe formation, and unsynchronized cellular growth between connective tissue and another lobe development. Analysis revealed arrested meiosis/cytokinesis in microspores, and altered lignin deposition pattern in endothecial walls thus affecting anther dehiscence. In the gynoecium, flaccid, dry stigmatic papillae, and large embryo sac in the female gametophyte was observed. Over-expression of miR159a thus severely affected pollination and seed-set. Analysis of the transcriptome data revealed components of regulatory networks of anther and carpel developmental pathway, and lignin metabolism that are affected. Expression analysis allowed us to position the miR159a-MYB65 module in the genetic network of stamen development, involved in pollen-grain maturation; in GA-mediated regulation of stamen development, and in lignin metabolism. The study, on one hand indicates role of miR159a-MYB65 in regulating multiple aspects of reproductive organ development that can be manipulated for trait modification, but also raises several unaddressed questions such as relationship between miR159a and male-meiosis, miR159a and filament elongation for future investigations. Accession numbers: KC204951-KC204960. Project number PRJNA1035268. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01377-7.

Combined genome-wide association study and epistasis analysis reveal multifaceted genetic architectures of plant height in Asian cultivated rice.

Plant height (PH) in rice (Oryza sativa) is an important trait for its adaptation and agricultural performance. Discovery of the semi-dwarf1 (SD1) mutation initiated the Green Revolution, boosting rice yield and fitness, but the underlying genetic regulation of PH in rice remains largely unknown. Here, we performed genome-wide association study (GWAS) and identified 12 non-repetitive QTL/genes regulating PH variation in 619 Asian cultivated rice accessions. One of these was an SD1 structural variant, not normally detected in standard GWAS analyses. Given the strong effect of SD1 on PH, we also divided 619 accessions into subgroups harbouring distinct SD1 haplotypes, and found a further 85 QTL/genes for PH, revealing genetic heterogeneity that may be missed by analysing a broad, diverse population. Moreover, we uncovered two epistatic interaction networks of PH-associated QTL/genes in the japonica (Geng)-dominant SD1NIP subgroup. In one of them, the hub QTL/gene qphSN1.4/GAMYB interacted with qphSN3.1/OsINO80, qphSN3.4/HD16/EL1, qphSN6.2/LOC_Os06g11130, and qphSN10.2/MADS56. Sequence variations in GAMYB and MADS56 were associated with their expression levels and PH variations, and MADS56 was shown to physically interact with MADS57 to coregulate expression of gibberellin (GA) metabolic genes OsGA2ox3 and Elongated Uppermost Internode1 (EUI1). Our study uncovered the multifaceted genetic architectures of rice PH, and provided novel and abundant genetic resources for breeding semi-dwarf rice and new candidates for further mechanistic studies on regulation of PH in rice.

Genome-Wide Comparative Analysis of R2R3 MYB Gene Family in Populus and Salix and Identification of Male Flower Bud Development-Related Genes.

The MYB transcription factor (TF) family is one of the largest plant transcription factor gene family playing vital roles in plant growth and development, including defense, cell differentiation, secondary metabolism, and responses to biotic and abiotic stresses. As a model tree species of woody plants, in recent years, the identification and functional prediction of certain MYB family members in the poplar genome have been reported. However, to date, the characterization of the gene family in the genome of the poplar's sister species willow has not been done, nor are the differences and similarities between the poplar and willow genomes understood. In this study, we conducted the first genome-wide investigation of the R2R3 MYB subfamily in the willow, identifying 216 R2R3 MYB gene members, and combined with the poplar R2R3 MYB genes, performed the first comparative analysis of R2R3 MYB genes between the poplar and willow. We identified 81 and 86 pairs of R2R3 MYB paralogs in the poplar and willow, respectively. There were 17 pairs of tandem repeat genes in the willow, indicating active duplication of willow R2R3 MYB genes. A further 166 pairs of poplar and willow orthologs were identified by collinear and synonymous analysis. The findings support the duplication of R2R3 MYB genes in the ancestral species, with most of the R2R3 MYB genes being retained during the evolutionary process. The phylogenetic trees of the R2R3 MYB genes of 10 different species were drawn. The functions of the poplar and willow R2R3 MYB genes were predicted using reported functional groupings and clustering by OrthoFinder. Identified 5 subgroups in general expanded in woody species, three subgroups were predicted to be related to lignin synthesis, and we further speculate that the other two subgroups also play a role in wood formation. We analyzed the expression patterns of the GAMYB gene of subgroup 18 (S18) related to pollen development in the male flower buds of poplar and willow at different developmental stages by qRT-PCR. The results showed that the GAMYB gene was specifically expressed in the male flower bud from pollen formation to maturity, and that the expression first increased and then decreased. Both the specificity of tissue expression specificity and conservation indicated that GAMYB played an important role in pollen development in both poplar and willow and was an ideal candidate gene for the analysis of male flower development-related functions of the two species.

Far-Red Light-Induced Azolla filiculoides Symbiosis Sexual Reproduction: Responsive Transcripts of Symbiont Nostoc azollae Encode Transporters Whilst Those of the Fern Relate to the Angiosperm Floral Transition.

Water ferns of the genus Azolla and the filamentous cyanobacteria Nostoc azollae constitute a model symbiosis that enabled the colonization of the water surface with traits highly desirable for the development of more sustainable crops: their floating mats capture CO2 and fix N2 at high rates using light energy. Their mode of sexual reproduction is heterosporous. The regulation of the transition from the vegetative phase to the spore forming phase in ferns is largely unknown, yet a prerequisite for Azolla domestication, and of particular interest as ferns represent the sister lineage of seed plants. Sporocarps induced with far red light could be crossed so as to verify species attribution of strains from the Netherlands but not of the strain from the Anzali lagoon in Iran; the latter strain was assigned to a novel species cluster from South America. Red-dominated light suppresses the formation of dissemination stages in both gametophyte- and sporophyte-dominated lineages of plants, the response likely is a convergent ecological strategy to open fields. FR-responsive transcripts included those from MIKCC homologues of CMADS1 and miR319-controlled GAMYB transcription factors in the fern, transporters in N. azollae, and ycf2 in chloroplasts. Loci of conserved microRNA (miRNA) in the fern lineage included miR172, yet FR only induced miR529 and miR535, and reduced miR319 and miR159. Phylogenomic analyses of MIKCC TFs suggested that the control of flowering and flower organ specification may have originated from the diploid to haploid phase transition in the homosporous common ancestor of ferns and seed plants.

Rice transcription factor GAMYB modulates bHLH142 and is homeostatically regulated by TDR during anther tapetal and pollen development.

GIBBERELLIN MYB GENE (GAMYB), UNDEVELOPED TAPETUM1 (UDT1), TDR INTERACTING PROTEIN2 (TIP2/bHLH142), TAPETUM DEGENERATION RETARDATION (TDR), and ETERNAL TAPETUM 1/DELAYED TAPETUM DEGENERATION (EAT1/DTD) are important transcription factors that play a crucial role during pollen development in rice. This study demonstrates that bHLH142 acts downstream of UDT1 and GAMYB and works as a 'hub' in these two pollen pathways. We show that GAMYB modulates bHLH142 expression through specific binding to the MYB motif of the bHLH142 promoter during the early stage of pollen development, while TDR acts as a transcriptional repressor of the GAMYB modulation of bHLH142 by binding to the E-box close to the MYB motif on the promoter. Altered expression of these transcription factors highlights that a tight, precise, and coordinated regulation among them is essential for normal pollen development. Most notably, we show that the regulatory pathways of GAMYB and UDT1 rely on bHLH142 in a direct and indirect manner, respectively, and function in different tissues with distinct biological roles during pollen development. This study advances our understanding of the molecular mechanisms of rice pollen development.

MiR319-targeted OsTCP21 and OsGAmyb regulate tillering and grain yield in rice.

Multiple genes and microRNAs (miRNAs) improve grain yield by promoting tillering. MiR319s are known to regulate several aspects of plant development; however, whether miR319s are essential for tillering regulation remains unclear. Here, we report that miR319 is highly expressed in the basal part of rice plant at different development stages. The miR319 knockdown line Short Tandem Target Mimic 319 (STTM319) showed higher tiller bud length in seedlings under low nitrogen (N) condition and higher tiller bud number under high N condition compared with the miR319a-overexpression line. Through targets prediction, we identified OsTCP21 and OsGAmyb as downstream targets of miR319. Moreover, OsTCP21 and OsGAmyb overexpression lines and STTM319 had increased tiller bud length and biomass, whereas both were decreased in OsTCP21 and OsGAmyb knockout lines and OE319a. These data suggest that miR319 regulates rice tiller bud development and tillering through targeting OsTCP21 and OsGAmyb. Notably, the tiller number and grain yield increased in STTM319 and overexpression lines of OsTCP21 and OsGAmyb but decreased in OE319a and knockout lines of OsTCP21 and OsGAmyb. Taken together, our findings indicate that miR319s negatively affect tiller number and grain yield by targeting OsTCP21 and OsGAmyb, revealing a novel function for miR319 in rice.

OsTPP1 regulates seed germination through the crosstalk with abscisic acid in rice.

Seed germination is essential for direct seeding in rice. It has been demonstrated that trehalose-6-phosphate phosphatase 1 (OsTPP1) plays roles in improving yield and stress tolerance in rice. In this study, the roles of OsTPP1 on seed germination in rice were investigated. The tpp1 mutant germinated slower than the wild-type (WT), which can be restored by exogenous trehalose. tpp1 seeds showed higher ABA content compared with WT seeds. The tpp1 mutant was hypersensitive to ABA and ABA catabolism inhibitor (Dinicozanole). Furthermore, two ABA catabolism genes were downregulated in the tpp1 mutant which were responsible for increased ABA concentrations, and exogenous trehalose increased transcripts of ABA catabolism genes, suggesting that OsTPP1 and ABA catabolism genes acted in the same signaling pathway. Further analysis showed that a transcription factor of OsGAMYB was an activator of OsTPP1, and expression of OsGAMYB was decreased by both the exogenous and endogenous ABA, subsequently reducing the expression of OsTPP1, which suggested a new signaling pathway required for seed germination in rice. In addition, ABA-responsive genes, especially OsABI5, were invoved in OsTPP1-mediated seed germination. Overall, our study provided new pathways in seed germination that OsTPP1 controlled seed germination through crosstalk with the ABA catabolism pathway.

The Crosstalk between MicroRNAs and Gibberellin Signaling in Plants.

Gibberellin (GA) is an integral phytohormone that plays prominent roles in controlling seed germination, stem elongation, leaf development and floral induction. It has been shown that GA regulates these diverse biological processes mainly through overcoming the suppressive effects of the DELLA proteins, a family of nuclear repressors of GA response. MicroRNAs (miRNAs), which have been identified as master regulators of gene expression in eukaryotes, are also involved in a wide range of plant developmental events through the repression of their target genes. The pathways of GA biosynthesis and signaling, as well as the pathways of miRNA biogenesis and regulation, have been profoundly delineated in the past several decades. Growing evidence has shown that miRNAs and GAs are coordinated in regulating plant development, as several components in GA pathways are targeted by miRNAs, and GAs also regulate the expression of miRNAs or their target genes vice versa. Here, we review the recent advances in our understanding of the molecular connections between miRNAs and GA, with an emphasis on the two miRNAs, miR156 and miR159.

Cloning and Functional Analysis of BcMYB101 Gene Involved in Leaf Development in Pak Choi (Brassica rapa ssp. Chinensis).

As one of the largest transcription factor families, MYB transcription factors are widely present, and they are involved in a diverse range of physiological activities in plants, such as leaf development. GAMYB genes belong to the R2R3-MYB subfamily, which includes the MYB33/65/101 gene, and these genes are studied well in seed germination and flowering, but their roles in leaf development are poorly understood. In the current study, we isolated a GAMYB transcription factor from pak choi, BcMYB101, and analyzed its characteristics and function. The sequence structure analysis indicated that BcMYB101 has a highly conserved R2R3 DNA-binding domain in the N-terminal region and three GAMYB-specific motifs (Box1, Box2, and Box3). The expression pattern of diverse tissues revealed that BcMYB101 has a higher transcript level in the petiole, leaf, root, and floral organs. Furthermore, the expression level was significantly elevated after GA (gibberellin) treatment, suggesting that the BcMYB101 response was positively regulated by GA. Subcellular localization exhibited that BcMYB101 was only present in the nuclear region, consistent with the characterization of the transcription factor. The overexpression of BcMYB101 elucidated that BcMYB101 increased leaf number and resulted in downward-curling cauline leaves. Moreover, the virus-induced BcMYB101 silencing displayed that BcMYB101 is involved in the regulation of curly leaves. Furthermore, we discovered that BcMYB101 has two trans-activation activities and one interaction protein, BcTCH4, using a trans-activation activity assay and a yeast two-hybrid assay, respectively. In this study, we firstly isolated the BcMYB101 gene and explored its function in leaf development, thereby providing a solid foundation for further research on the regulatory mechanism of leaf shape in Brassica or other species.

The lre-miR159a-LrGAMYB pathway mediates resistance to grey mould infection in Lilium regale.

Grey mould is one of the most determinative factors of lily growth and plays a major role in limiting lily productivity. MicroRNA159 (miR159) is a highly conserved microRNA in plants, and participates in the regulation of plant development and stress responses. Our previous studies revealed that lre-miR159a participates in the response of Lilium regale to Botrytis elliptica according to deep sequencing analyses; however, the response mechanism remains unknown. Here, lre-miR159a and its target LrGAMYB gene were isolated from L. regale. Transgenic Arabidopsis overexpressing lre-MIR159a exhibited larger leaves and smaller necrotic spots on inoculation with Botrytis than those of wild-type and overexpressing LrGAMYB plants. The lre-MIR159a overexpression also led to repressed expression of two targets of miR159, AtMYB33 and AtMYB65, and enhanced accumulation of hormone-related genes, including AtPR1, AtPR2, AtNPR1, AtPDF1.2, and AtLOX for both the jasmonic acid and salicylic acid pathways. Moreover, lower levels of H2 O2 and O2- were observed in lre-MIR159a transgenic Arabidopsis, which reduced the damage from reactive oxygen species accumulation. Taken together, these results indicate that lre-miR159a positively regulates resistance to grey mould by repressing the expression of its target LrGAMYB gene and activating a defence response.

Biology and Function of miR159 in Plants.

MicroR159 (miR159) is ancient, being present in the majority of land plants where it targets a class of regulatory genes called GAMYB or GAMYB-like via highly conserved miR159-binding sites. These GAMYB genes encode R2R3 MYB domain transcription factors that transduce the gibberellin (GA) signal in the seed aleurone and the anther tapetum. Here, GAMYB plays a conserved role in promoting the programmed cell death of these tissues, where miR159 function appears weak. By contrast, GAMYB is not involved in GA-signaling in vegetative tissues, but rather its expression is deleterious, leading to the inhibition of growth and development. Here, the major function of miR159 is to mediate strong silencing of GAMYB to enable normal growth. Highlighting this requirement of strong silencing are conserved RNA secondary structures associated with the miR159-binding site in GAMYB mRNA that promotes miR159-mediated repression. Although the miR159-GAMYB pathway in vegetative tissues has been implicated in a number of different functions, presently no conserved role for this pathway has emerged. We will review the current knowledge of the different proposed functions of miR159, and how this ancient pathway has been used as a model to help form our understanding of miRNA biology in plants.

microRNA159-targeted SlGAMYB transcription factors are required for fruit set in tomato.

The transition from flowering to fruit production, namely fruit set, is crucial to ensure successful sexual plant reproduction. Although studies have described the importance of hormones (i.e. auxin and gibberellins) in controlling fruit set after pollination and fertilization, the role of microRNA-based regulation during ovary development and fruit set is still poorly understood. Here we show that the microRNA159/GAMYB1 and -2 pathway (the miR159/GAMYB1/2 module) is crucial for tomato ovule development and fruit set. MiR159 and SlGAMYBs were expressed in preanthesis ovaries, mainly in meristematic tissues, including developing ovules. SlMIR159-overexpressing tomato cv. Micro-Tom plants exhibited precocious fruit initiation and obligatory parthenocarpy, without modifying fruit shape. Histological analysis showed abnormal ovule development in such plants, which led to the formation of seedless fruits. SlGAMYB1/2 silencing in SlMIR159-overexpressing plants resulted in misregulation of pathways associated with ovule and female gametophyte development and auxin signalling, including AINTEGUMENTA-like genes and the miR167/SlARF8a module. Similarly to SlMIR159-overexpressing plants, SlGAMYB1 was downregulated in ovaries of parthenocarpic mutants with altered responses to gibberellins and auxin. SlGAMYBs likely contribute to fruit initiation by modulating auxin and gibberellin responses, rather than their levels, during ovule and ovary development. Altogether, our results unveil a novel function for the miR159-targeted SlGAMYBs in regulating an agronomically important trait, namely fruit set.

Ubiquitous miR159 repression of MYB33/65 in Arabidopsis rosettes is robust and is not perturbed by a wide range of stresses.

BACKGROUND: The microR159 (miR159) - GAMYB pathway is conserved in higher plants, where GAMYB, expression promotes programmed cell death in seeds (aleurone) and anthers (tapetum). In cereals, restriction of GAMYB expression to seeds and anthers is mainly achieved transcriptionally, whereas in Arabidopsis this is achieved post-transcriptionally, as miR159 silences GAMYB (MYB33 and MYB65) in vegetative tissues, but not in seeds and anthers. However, we cannot rule out a role for miR159-MYB33/65 pathway in Arabidopsis vegetative tissues; a loss-of-function mir159 Arabidopsis mutant displays strong pleiotropic defects and numerous reports have documented changes in miR159 abundance during stress and hormone treatments. Hence, we have investigated the functional role of this pathway in vegetative tissues. RESULTS: It was found that the miR159-MYB33/65 pathway was ubiquitously present throughout rosette development. However, miR159 appears to continuously repress MYB33/MYB65 expression to levels that have no major impact on rosette development. Inducible inhibition of miR159 resulted in MYB33/65 de-repression and associated phenotypic defects, indicating that a potential role in vegetative development is only possible through MYB33 and MYB65 if miR159 levels decrease. However, miR159 silencing of MYB33/65 appeared extremely robust; no tested abiotic stress resulted in strong miR159 repression. Consistent with this, the stress responses of an Arabidopsis mutant lacking the miR159-MYB33/65 pathway were indistinguishable from wild-type. Moreover, expression of viral silencing suppressors, either via transgenesis or viral infection, was unable to prevent miR159 repression of MYB33/65, highlighting the robustness of miR159-mediated silencing. CONCLUSIONS: Despite being ubiquitously present, molecular, genetic and physiological analysis failed to find a major functional role for the miR159-MYB33/65 pathway in Arabidopsis rosette development or stress response. Although it is likely that this pathway is important for a stress not tested here or in different plant species, our findings argue against the miR159-MYB33/65 pathway playing a major conserved role in general stress response. Finally, in light of the robustness of miR159-mediated repression of MYB33/65, it appears unlikely that low fold-level changes of miR159 abundance in response to stress would have any major physiological impact in Arabidopsis.

Identification of miR159s and their target genes and expression analysis under drought stress in potato.

The MYB proteins comprise one of the largest families of plant transcription factors (TFs) and many of MYB families, which play essential roles in plant growth, development and respond to environmental stresses, and have yet been identified in plant. Previous research has shown that miR159 family members repressed the conserved plant R2R3 MYB domain TFs in model plants. In the present research, we identified three potato novel miR159 family members named as stu-miR159a, stu-miR159b and stu-miR159c based on bioinformatics analysis. Target prediction showed that they have a bite sit on the three GAMyb-like genes (StGAMyb-like1, StGAMyb-like2.1 and StGAMyb-like2.2) of potato. Those GAMyb-like genes also have been selected and cloned from potato, which belong to R2R3 MYB domain TFs. We further measured expressional levels of stu-miR159s and potato GAMyb-like genes during the different periods of drought treated samples using quantitative real-time PCR (qRT-PCR). The results showed that they had a opposite expression pattern, briefly, three stu-miR159 members showed similar expressional trends which were significantly decreased expression after experiencing 25 days of drought stress treatment, while the potato GAMyb-like family members were greatly increased. Therefore, we suggested that stu-miR159s negatively regulated the expression of potato GAMyb-like genes which responsible for drought stress. The findings can facilitate functional studies of miRNAs in plants and provide molecular evidence for involvement process of drought tolerance in potato.

A GAMYB homologue CsGAMYB1 regulates sex expression of cucumber via an ethylene-independent pathway.

Cucumber (Cucumis sativus L.) is a typical monoecious vegetable with individual male and female flowers, and has been used as a model plant for sex determination. It is well known that sex differentiation of cucumber can be regulated by phytohormones, such as gibberellic acid (GA) and ethylene. The molecular mechanism of female sex expression modulated by ethylene has been widely understood, but how GA controls male sex expression remains elusive. In hermaphroditic Arabidopsis and rice, GA can regulate stamen and anther development via the transcriptional regulation of GAMYB. Here we characterized a GAMYB homologue CsGAMYB1 in cucumber. We found that CsGAMYB1 is predominantly expressed in male flower buds, where its expression is upregulated by GA3 treatment. CsGAMYB1 protein is localized in the nucleus. CsGAMYB1 can partially rescue stamen development and fertility phenotypes of an Arabidopsis myb33 myb65 double mutant. However, constitutive overexpression of CsGAMYB1 in wild-type Arabidopsis resulted in male sterility, which mimics the effect of GA overdose in flower development. Knockdown of CsGAMYB1 in cucumber decreases the ratio of nodes with male and female flowers, and ethylene is not involved in this process. Our data suggest that CsGAMYB1 regulates sex expression of cucumber via an ethylene-independent pathway.