BBX21 integrates brassinosteroid biosynthesis and signalling in the inhibition of hypocotyl growth under shade.

B-Box-containing zinc finger transcription factors (BBX) are involved in light-mediated growth, affecting processes such as hypocotyl elongation in Arabidopsis thaliana. However, the molecular and hormonal framework that regulates plant growth through BBX proteins is incomplete. Here, we demonstrate that BBX21 inhibits the hypocotyl elongation through the brassinosteroid (BR) pathway. BBX21 reduces the sensitivity to 24-epiBL, a synthetic active BR, principally at very-low concentrations in simulated shade. The biosynthesis profile of BRs showed that two active BR -brassinolide (BL) and 28-homobrassinolide (28-homoBL)- and 8 of 11 intermediates can be repressed by BBX21 under white light (WL) or simulated shade. Furthermore, BBX21 represses the expression of CYTOCHROME P450 90B1 (DWF4/CYP90B1), BRASSINOSTEROID-6-OXIDASE 1 (BR6OX1, CYP85A1) and BR6OX2 (CYP85A2) genes involved in the BR biosynthesis in WL while specifically promoting DWF4 and PHYB ACTIVATION TAGGED SUPPRESSOR 1 (CYP2B1/BAS1) expression in WL supplemented with far-red (WL+FR), a treatment that simulates shade. In addition, BBX21 represses BR signalling genes such as PACLOBUTRAZOL RESISTANCE1 (PRE1), PRE3 and ARABIDOPSIS MYB-LIKE 2 (MYBL2), and auxin-related and expansin genes, such as INDOLE-3-ACETIC ACID INDUCIBLE 1 (IAA1), IAA4 and EXPANSIN 11 (EXP11) in short-term shade. By a genetic approach we found that BBX21 acts genetically upstream of BRASSINAZOLE-RESISTANT 1 (BZR1) for the promotion of DWF4 and BAS1 gene expression in shade. We propose that BBX21 integrates the BR homeostasis and shade-light signalling allowing the fine-tuning of hypocotyl elongation in Arabidopsis.

Transcriptomic and physiological shade avoidance responses in potato (Solanum tuberosum) plants.

Plants detect competitors in shaded environments by perceiving a reduction in photosynthetically active radiation (PAR) and the reduction between the red and far-red light (R:FR) ratio and blue photons. These light signals are detected by phytochromes and cryptochromes, which trigger shade avoidance responses such as shoot and petiole elongation and lead to increased susceptibility to pathogen attack. We studied morphological, anatomical, and photosynthesis differences in potato plants (Solanum tuberosum var. Spunta) exposed to sunlight or simulated shade in a greenhouse. We found that simulated shade strongly induced stem and internode elongation with a higher production of free auxin in stems and a lower production of tubers. The mesophyll thickness of the upper leaves of plants grown in simulated shade was lower, but the epidermis was wider compared with the leaves of plants cultivated in sunlight. In addition, the photosynthesis rate was lower in the upper leaves exposed to nonsaturated irradiances and higher in the basal leaves at saturated irradiances compared with control plants. RNA-seq analysis showed that 146 and 155 genes were up- and downregulated by shade, respectively. By quantitative reverse transcription polymerase chain reaction, we confirmed that FLOWERING LOCUS T (FT), WRKY-like, and PAR1b were induced, while FLAVONOL 4-SULFOTRANSFERASE was repressed under shade. In shaded plants, leaves and tubers were more susceptible to the necrotrophic fungus Botrytis cinerea attack. Overall, our work demonstrates configurational changes between growth and defense decisions in potato plants cultivated in simulated shade.

BBX24 Interacts with JAZ3 to Promote Growth by Reducing DELLA Activity in Shade Avoidance.

Shade avoidance syndrome (SAS) is a strategy of major adaptive significance and typically includes elongation of the stem and petiole, leaf hyponasty, reduced branching and phototropic orientation of the plant shoot toward canopy gaps. Both cryptochrome 1 and phytochrome B (phyB) are the major photoreceptors that sense the reduction in the blue light fluence rate and the low red:far-red ratio, respectively, and both light signals are associated with plant density and the resource reallocation when SAS responses are triggered. The B-box (BBX)-containing zinc finger transcription factor BBX24 has been implicated in the SAS as a regulator of DELLA activity, but this interaction does not explain all the observed BBX24-dependent regulation in shade light. Here, through a combination of transcriptional meta-analysis and large-scale identification of BBX24-interacting transcription factors, we found that JAZ3, a jasmonic acid signaling component, is a direct target of BBX24. Furthermore, we demonstrated that joint loss of BBX24 and JAZ3 function causes insensitivity to DELLA accumulation, and the defective shade-induced elongation in this mutant is rescued by loss of DELLA or phyB function. Therefore, we propose that JAZ3 is part of the regulatory network that controls the plant growth in response to shade, through a mechanism in which BBX24 and JAZ3 jointly regulate DELLA activity. Our results provide new insights into the participation of BBX24 and JA signaling in the hypocotyl shade avoidance response in Arabidopsis.

Functional analysis of PHYB polymorphisms in Arabidopsis thaliana collected in Patagonia.

Arabidopsis thaliana shows a wide range of natural genetic variation in light responses. Shade avoidance syndrome is a strategy of major adaptive significance that includes seed germination, elongation of vegetative structures, leaf hyponasty, and acceleration of flowering. Previously, we found that the southernmost Arabidopsis accession, collected in the south of Patagonia (Pat), is hyposensitive to light and displays a reduced response to shade light. This work aimed to explore the genetic basis of the shade avoidance response (SAR) for hypocotyl growth by QTL mapping in a recently developed 162 RIL population between Col-0 and Pat. We mapped four QTL for seedling hypocotyl growth: WL1 and WL2 QTL in white light, SHADE1 QTL in shade light, and SAR1 QTL for the SAR. PHYB is the strongest candidate gene for SAR1 QTL. Here we studied the function of two polymorphic indels in the promoter region, a GGGR deletion, and three non-synonymous polymorphisms on the PHYB coding region compared with the Col-0 reference genome. To decipher the contribution and relevance of each PHYB-Pat polymorphism, we constructed transgenic lines with single or double polymorphisms by using Col-0 as a reference genome. We found that single polymorphisms in the coding region of PHYB have discrete functions in seed germination, seedling development, and shade avoidance response. These results suggest distinct functions for each PHYB polymorphism to the adjustment of plant development to variable light conditions.

Differential DNA methylation and gene expression during development of reproductive and vegetative organs in Ilex species.

Differential epigenetic (DNA cytosine methylation) and gene expression patterns were investigated in reproductive and vegetative organs from Ilex paraguariensis and I. dumosa, at distinct developmental stages. We aimed at contributing towards elucidating major molecular changes underlying the sexual differentiation processes which, in these dioecious species, are completely unknown. Simultaneously, as a first step towards the development of an early sexing system, we searched for promising molecular markers. This was assessed through Methylation Sensitive Amplified Polymorphism (MSAP) and Amplified Fragment Length Polymorphism on cDNA (cDNA-AFLP) techniques, applying discriminant multivariate analyses, and bioinformatic characterization of differential fragments. A significant positive correlation was found between epigenetic and indirect 'genetic' information for both species, indicating influence of the genetic background on the epigenetic variation. Higher epigenetic than genetic diversities were estimated. Our outcomes showed up to 1.86 times more representation of mCG subepiloci than mCCG in all organs sampled. Along the maturing stages of floral buds, the frequency of mCG evidenced an incremental trend, whereas mCCG and unmethylated conditions showed opposite tendencies. Reproductive and vegetative samples tended to cluster apart based on epigenetic patterns; at gene expression level, organs exhibited clear-cut distinctive patterns, nonetheless profiles of young leaves and floral primordia resemble. Epigenetic and expression data allowed discrimination of I. dumosa´s samples according to the gender of the donor; more elusive patterns were observed for I. paraguariensis. In total, 102 differentially methylated and expressed fragments were characterized bioinformatically. Forty-three were annotated in various functional categories; four candidate markers were validated through qPCR, finding statistical differences among organs but not among sexes. The methylation condition of epilocus C13m33 appears as indicative of gender in both species. Thirty-three organ-specific and 34 gender-specific methylated markers were discriminated and deserve further research, particularly those expressed in leaves. Our study contributes concrete candidate markers with potential for practical application.

Unraveling the genetic complexity of a cultivated breeding population of "yerba mate" (Ilex paraguariensis St. Hil.).

We report the first comprehensive multilocus molecular characterization of cultivated Ilex paraguariensis plants belonging to a breeding program. Using nuclear and homologous chloroplastidic molecular markers, we have genotyped 158 plants from four plantation sites. Analyses of the nuclear data (187 variable dominant loci) allowed detecting high diversity (0.569), the occurrence of four distinct genetic clusters, and a low but significant differentiation among sites. Additionally, 20 chloroplastidic alleles were identified applying five microsatellite polymorphic markers, and a high chloroplastidic diversity was recognized (0.505); two haplogroups were distinguished amongst the 63 haplotypes detected. Our results from both nuclear and plastidic markers indicate that most genetic variation reside within plantations sites (≥ 95%), and that these plantations were established on highly variable materials (either as seeds or plantlets) derived from, at least, 63 maternal lineages. Moreover, our study suggests that the genetic structure within each plantation site was most likely shaped by past admixture favored by farmers´ practices during the establishment of each plantation. Also, subsequent constraints in gene flow and/or a low level of shared polymorphism among plantations could have contributed to current structure.

Integral Phylogenomic Approach over Ilex L. Species from Southern South America.

The use of molecular markers with inadequate variation levels has resulted in poorly resolved phylogenetic relationships within Ilex. Focusing on southern South American and Asian species, we aimed at contributing informative plastid markers. Also, we intended to gain insights into the nature of morphological and physiological characters used to identify species. We obtained the chloroplast genomes of I.paraguariensis and I. dumosa, and combined these with all the congeneric plastomes currently available to accomplish interspecific comparisons and multilocus analyses. We selected seven introns and nine IGSs as variable non-coding markers that were used in phylogenomic analyses. Eight extra IGSs were proposed as candidate markers. Southern South American species formed one lineage, except for I. paraguariensis, I. dumosa and I. argentina, which occupied intermediate positions among sampled taxa; Euroasiatic species formed two lineages. Some concordant relationships were retrieved from nuclear sequence data. We also conducted integral analyses, involving a supernetwork of molecular data, and a simultaneous analysis of quantitative and qualitative morphological and phytochemical characters, together with molecular data. The total evidence tree was used to study the evolution of non-molecular data, evidencing fifteen non-ambiguous synapomorphic character states and consolidating the relationships among southern South American species. More South American representatives should be incorporated to elucidate their origin.

Dissecting maize diversity in lowland South America: genetic structure and geographic distribution models.

BACKGROUND: Maize landraces from South America have traditionally been assigned to two main categories: Andean and Tropical Lowland germplasm. However, the genetic structure and affiliations of the lowland gene pools have been difficult to assess due to limited sampling and the lack of comparative analysis. Here, we examined SSR and Adh2 sequence variation in a diverse sample of maize landraces from lowland middle South America, and performed a comprehensive integrative analysis of population structure and diversity including already published data of archaeological and extant specimens from the Americas. Geographic distribution models were used to explore the relationship between environmental factors and the observed genetic structure. RESULTS: Bayesian and multivariate analyses of population structure showed the existence of two previously overlooked lowland gene pools associated with Guaraní indigenous communities of middle South America. The singularity of this germplasm was also evidenced by the frequency distribution of microsatellite repeat motifs of the Adh2 locus and the distinct spatial pattern inferred from geographic distribution models. CONCLUSION: Our results challenge the prevailing view that lowland middle South America is just a contact zone between Andean and Tropical Lowland germplasm and highlight the occurrence of a unique, locally adapted gene pool. This information is relevant for the conservation and utilization of maize genetic resources, as well as for a better understanding of environment-genotype associations.

Genetic diversity of wild germplasm of "yerba mate" (Ilex paraguariensis St. Hil.) from Uruguay.

The "yerba mate" tree, Ilex paraguariensis St. Hil., is a crop native to subtropical South America, marketed for the elaboration of the highly popular "mate" beverage. The Uruguayan germplasm occupies the southernmost area of the species distribution range and carries adaptations to environments that considerably differ from the current production area. We characterized the genetic variability of the germplasm from this unexplored area by jointly analyzing individuals from the diversification center (ABP, Argentina, Brazil and Paraguay) with 19 nuclear and 11 plastidic microsatellite markers. For the Uruguayan germplasm, we registered 55 alleles (18 % private), and 80 genotypes (44 % exclusive), whereas 63 alleles (28.6 % private) and 81 genotypes (42 % exclusive) were recorded for individuals from ABP. Only two plastidic haplotypes were detected. Distance-based and multilocus genotype analyses showed that individuals from ABP intermingle and that the Uruguayan germplasm is differentiated in three gene-pools. Significant positive correlations between genetic and geographic distances were detected. Our results concur in that ABP individuals harbor greater genetic variation than those from the tail of the distribution, as to the number of alleles (1.15-fold), He (1.19-fold), Rs (1.39-fold), and the between-group genetic distances (1.16-fold). Also the shape of the genetic landscape interpolation analysis suggests that the genetic variation decays southward towards the Uruguayan territory. We showed that Uruguayan germplasm hosts a combination of nuclear alleles not present in the central region, constituting a valuable breeding resource. Future conservation efforts should concentrate in collecting numerous individuals of "yerba mate" per site to gather the existent variation.