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.

AtBBX29 integrates photomorphogenesis and defense responses in Arabidopsis.

Light is an environmental signal that modulates plant defenses against attackers. Recent research has focused on the effects of light on defense hormone signaling; however, the connections between light signaling pathways and the biosynthesis of specialized metabolites involved in plant defense have been relatively unexplored. Here, we show that Arabidopsis BBX29, a protein that belongs to the B-Box transcription factor (TF) family, integrates photomorphogenic signaling with defense responses by promoting flavonoid, sinapate and glucosinolate accumulation in Arabidopsis leaves. AtBBX29 transcript levels were up regulated by light, through photoreceptor signaling pathways. Genetic evidence indicated that AtBBX29 up-regulates MYB12 gene expression, a TF known to induce genes related to flavonoid biosynthesis in a light-dependent manner, and MYB34 and MYB51, which encode TFs involved in the regulation of glucosinolate biosynthesis. Thus, bbx29 knockout mutants displayed low expression levels of key genes of the flavonoid biosynthetic pathway, and the opposite was true in BBX29 overexpression lines. In agreement with the transcriptomic data, bbx29 mutant plants accumulated lower levels of kaempferol glucosides, sinapoyl malate, indol-3-ylmethyl glucosinolate (I3M), 4-methylsulfinylbutyl glucosinolate (4MSOB) and 3-methylthiopropyl glucosinolate (3MSP) in rosette leaves compared to the wild-type, and showed increased susceptibility to the necrotrophic fungus Botrytis cinerea and to the herbivore Spodoptera frugiperda. In contrast, BBX29 overexpressing plants displayed increased resistance to both attackers. In addition, we found that AtBBX29 plays an important role in mediating the effects of ultraviolet-B (UV-B) radiation on plant defense against B. cinerea. Taken together, these results suggest that AtBBX29 orchestrates the accumulation of specific light-induced metabolites and regulates Arabidopsis resistance against pathogens and herbivores.

BBX21 reduces abscisic acid sensitivity, mesophyll conductance and chloroplast electron transport capacity to increase photosynthesis and water use efficiency in potato plants cultivated under moderated drought.

The B-box (BBX) proteins are zinc-finger transcription factors with a key role in growth and developmental regulatory networks mediated by light. AtBBX21 overexpressing (BBX21-OE) potato (Solanum tuberosum) plants, cultivated in optimal water conditions, have a higher photosynthesis rate and stomatal conductance without penalty in water use efficiency (WUE) and with a higher tuber yield. In this work, we cultivated potato plants in two water regimes: 100 and 35% field capacity of water restriction that imposed leaf water potentials between -0.3 and -1.2 MPa for vegetative and tuber growth during 14 or 28 days, respectively. We found that 42-day-old plants of BBX21-OE were more tolerant to water restriction with higher levels of chlorophylls and tuber yield than wild-type spunta (WT) plants. In addition, the BBX21-OE lines showed higher photosynthesis rates and WUE under water restriction during the morning. Mechanistically, we found that BBX21-OE lines were more tolerant to moderated drought by enhancing mesophyll conductance (gm ) and maximum capacity of electron transport (Jmax ), and by reducing abscisic acid (ABA) sensitivity in plant tissues. By RNA-seq analysis, we found 204 genes whose expression decreased by drought in WT plants and expressed independently of the water condition in BBX21-OE lines as SAP12, MYB73, EGYP1, TIP2-1 and DREB2A, and expressions were confirmed by quantitative polymerase chain reaction. These results suggest that BBX21 interplays with the ABA and growth signaling networks, improving the photosynthetic behavior in suboptimal water conditions with an increase in potato tuber yield.

Setting Up an Arabidopsis LED Culture Module that Simulates Plant Neighbor Proximity.

Competition for light between neighboring plants has important consequences for plant fitness and crop productivity. Studies on the molecular mechanisms of plant responses to neighbor proximity have been largely based on the model species Arabidopsis thaliana grown under controlled light environments. These controlled conditions commonly use fluorescent tubes for the main light source for photosynthesis and filtered light form incandescent bulbs to adjust the ratio of red (R) to far-red (FR) radiation. However, both of these types of bulbs are being discontinued and replaced by more efficient sources based on light emitting diodes (LEDs). For that reason, there is a need to evaluate alternative light sources, which can phenocopy the physiological and molecular results obtained with traditional lighting systems. Here we evaluate a custom-made LED culture module that can be used to effectively evaluate shade-avoidance responses, yielding results that, in Arabidopsis, are comparable to those obtained using traditional lighting systems.

Maternal Exposure to Ozone Modulates the Endophyte-Conferred Resistance to Aphids in Lolium multiflorum Plants.

Plants are challenged by biotic and abiotic stress factors and the incidence of one can increase or decrease resistance to another. These relations can also occur transgenerationally. For instance, progeny plants whose mothers experienced herbivory can be more resistant to herbivores. Certain fungal endophytes that are vertically transmitted endow plants with alkaloids and resistance to herbivores. However, endophyte-symbiotic plants exposed to the oxidative agent ozone became susceptible to aphids. Here, we explored whether this effect persists transgenerationally. We exposed Lolium multiflorum plants with and without fungal endophyte Epichloë occultans to ozone (120 or 0 ppb), and then, challenged the progeny with aphids (Rhopalosiphum padi). The endophyte was the main factor determining the resistance to aphids, but its importance diminished in plants with ozone history. This negative ozone effect on the endophyte-mediated resistance was apparent on aphid individual weights. Phenolic compounds in seeds were increased by the symbiosis and diminished by the ozone. The endophyte effect on phenolics vanished in progeny plants while the negative ozone effect persisted. Independently of ozone, the symbiosis increased the plant biomass (≈24%). Although ozone can diminish the importance of endophyte symbiosis for plant resistance to herbivores, it would be compensated by host growth stimulation.

A light-dependent molecular link between competition cues and defence responses in plants.

Growth responses to competition1 and defence responses to the attack of consumer organisms2 are two classic examples of adaptive phenotypic plasticity in plants. However, the mechanistic and functional links between these responses are not well understood. Jasmonates, a family of lipid-derived signals, are potent growth inhibitors and central regulators of plant immunity to herbivores and pathogens3,4, with both roles being evolutionarily conserved from bryophytes5 to angiosperms6. When shade-intolerant plants perceive the proximity of competitors using the photoreceptor phytochrome B, they activate the shade-avoidance syndrome and downregulate jasmonate responses7. Despite the central implications of this light-mediated change in the growth/defence balance for plant adaptation and crop yield8,9, the mechanisms by which photoreceptors relay light cues to the jasmonate signalling pathway remain poorly understood10. Here, we identify a sulfotransferase (ST2a) that is strongly upregulated by plant proximity perceived by phytochrome B via the phytochrome B-phytochrome interacting factor signalling module. By catalysing the formation of a sulfated jasmonate derivative, ST2a acts to reduce the pool of precursors of active forms of jasmonates and represents a direct molecular link between photoreceptors and hormone signalling in plants. The metabolic step defined by this enzyme provides a molecular mechanism for prioritizing shade avoidance over defence under intense plant competition.

Heterologous Expression of AtBBX21 Enhances the Rate of Photosynthesis and Alleviates Photoinhibition in Solanumtuberosum.

B-box (BBX) proteins are zinc-finger transcription factors containing one or two B-box motifs. BBX proteins act as key factors in the networks regulating growth and development. The relevance of BBX21 to light and abscisic acid signaling in seedling development is well established; however, its importance in adult plant development and agronomic species is poorly understood. Therefore, we studied the effect of heterologous expression of Arabidopsis (Arabidopsis thaliana) BBX21 in potato (Solanum tuberosum) var Spunta. Three independent AtBBX21-expressing lines and the wild-type control were cultivated under sunlight and at controlled temperatures in a greenhouse. By anatomical, physiological, biochemical, and gene expression analysis, we demonstrated that AtBBX21-expressing plants were more robust and produced more tubers than wild-type plants. Interestingly, AtBBX21-expressing plants had higher rates of photosynthesis, with a significant increase in photosynthetic gene expression, and higher stomatal conductance, with increased size of the stomatal opening, without any associated decline in water use efficiency. Furthermore, AtBBX21-expressing potato plants had reduced photoinhibition associated with higher production of anthocyanins and phenolic compounds, and higher expression of genes in the phenylpropanoid biosynthesis pathway. To gain insights into the mechanism of BBX21, we evaluated the molecular, morphological, metabolic, and photosynthetic behavior in adult BBX21-overexpressing Arabidopsis. We conclude that BBX21 overexpression improved morphological and physiological attributes, and photosynthetic rates in nonoptimal, high-irradiance conditions, without associated impairment of water use efficiency. These characteristics of BBX21 may be useful for increasing production of potatoes, and potentially of other crops.

UV-B Perception and Acclimation in Chlamydomonas reinhardtii.

Plants perceive UV-B, an intrinsic component of sunlight, via a signaling pathway that is mediated by the photoreceptor UV RESISTANCE LOCUS8 (UVR8) and induces UV-B acclimation. To test whether similar UV-B perception mechanisms exist in the evolutionarily distant green alga Chlamydomonas reinhardtii, we identified Chlamydomonas orthologs of UVR8 and the key signaling factor CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1). Cr-UVR8 shares sequence and structural similarity to Arabidopsis thaliana UVR8, has conserved tryptophan residues for UV-B photoreception, monomerizes upon UV-B exposure, and interacts with Cr-COP1 in a UV-B-dependent manner. Moreover, Cr-UVR8 can interact with At-COP1 and complement the Arabidopsis uvr8 mutant, demonstrating that it is a functional UV-B photoreceptor. Chlamydomonas shows apparent UV-B acclimation in colony survival and photosynthetic efficiency assays. UV-B exposure, at low levels that induce acclimation, led to broad changes in the Chlamydomonas transcriptome, including in genes related to photosynthesis. Impaired UV-B-induced activation in the Cr-COP1 mutant hit1 indicates that UVR8-COP1 signaling induces transcriptome changes in response to UV-B. Also, hit1 mutants are impaired in UV-B acclimation. Chlamydomonas UV-B acclimation preserved the photosystem II core proteins D1 and D2 under UV-B stress, which mitigated UV-B-induced photoinhibition. These findings highlight the early evolution of UVR8 photoreceptor signaling in the green lineage to induce UV-B acclimation and protection.

Revisiting chromatin binding of the Arabidopsis UV-B photoreceptor UVR8.

BACKGROUND: Plants perceive UV-B through the UV RESISTANCE LOCUS 8 (UVR8) photoreceptor and UVR8 activation leads to changes in gene expression such as those associated with UV-B acclimation and stress tolerance. Albeit functionally unrelated, UVR8 shows some homology with RCC1 (Regulator of Chromatin Condensation 1) proteins from non-plant organisms at the sequence level. These proteins act as guanine nucleotide exchange factors for Ran GTPases and bind chromatin via histones. Subsequent to the revelation of this sequence homology, evidence was presented showing that UVR8 activity involves interaction with chromatin at the loci of some target genes through histone binding. This suggested a UVR8 mode-of-action intimately and directly linked with gene transcription. However, several aspects of UVR8 chromatin association remained undefined, namely the impact of UV-B on the process and how UVR8 chromatin association related to the transcription factor ELONGATED HYPOCOTYL 5 (HY5), which is important for UV-B signalling and has overlapping chromatin targets. Therefore, we have investigated UVR8 chromatin association in further detail. RESULTS: Unlike the claims of previous studies, our chromatin immunoprecipitation (ChIP) experiments do not confirm UVR8 chromatin association. In contrast to human RCC1, recombinant UVR8 also does not bind nucleosomes in vitro. Moreover, fusion of a VP16 activation domain to UVR8 did not alter expression of proposed UVR8 target genes in transient gene expression assays. Finally, comparison of the Drosophila DmRCC1 and the Arabidopsis UVR8 crystal structures revealed that critical histone- and DNA-interaction residues apparent in DmRCC1 are not conserved in UVR8. CONCLUSION: This has led us to conclude that the cellular activity of UVR8 likely does not involve its specific binding to chromatin at target genes.

The transcriptional regulator BBX24 impairs DELLA activity to promote shade avoidance in Arabidopsis thaliana.

In response to canopy shade, plant vegetative structures elongate to gain access to light. However, the mechanism that allows a plastic transcriptional response to canopy shade light is not fully elucidated. Here we propose that the activity of PIF4, a key transcription factor in the shade signalling network, is modulated by the interplay between the BBX24 transcriptional regulator and DELLA proteins, which are negative regulators of the gibberellin (GA) signalling pathway. We show that GA-related targets are enriched among genes responsive to BBX24 under shade and that the shade-response defect in bbx24 mutants is rescued by a GA treatment that promotes DELLA degradation. BBX24 physically interacts with DELLA proteins and alleviates DELLA-mediated repression of PIF4 activity. The proposed molecular mechanism provides reversible regulation of the activity of a key transcription factor that may prove especially relevant under fluctuating light conditions.

BBX proteins in green plants: insights into their evolution, structure, feature and functional diversification.

The B-box domain is conserved in a large number of proteins involved in cell growth control, differentiation and transcriptional regulation among animal and plant species. In Arabidopsis thaliana, some works have found that B-box proteins (BBX) play central developmental functions in flowering, light and abiotic stress signaling. Despite the functional importance of this protein family, evolutionary and structural relationships of BBX proteins have not been extensively investigated in the plant kingdom. Using a phylogenetic approach, we conducted a comprehensive evolutionary analysis of the BBX protein family in twelve plant species (four green algae, one moss, one lycophyte, three monocots and three dicots). The analysis classified 214 BBX proteins into five structure groups, which evolved independently at early stages of green plant evolution. We showed that the B-box consensus sequences of each structure groups retained a common and conserved domain topology. Furthermore, we identified seven novel motifs specific to each structure group and a valine-proline (VP) pair conserved at the C-terminus domain in some BBX proteins suggesting that they are required for protein-protein interactions. As it has been documented in mammalian systems, we also found monopartite and bipartite amino acid sequences at the C-terminus domain that could function as nuclear localization signals (NLSs). The five BBX structure groups evolved constrained by the conservation of amino acid sequences in the two B-boxes, but radiating variation into NLSs and novel motifs of each structural group. We suggest that these features are the functional basis for the BBX protein diversity in green plants.

The Arabidopsis B-BOX protein BBX25 interacts with HY5, negatively regulating BBX22 expression to suppress seedling photomorphogenesis.

ELONGATED HYPOCOTYL5 (HY5) is a basic domain/leucine zipper (bZIP) transcription factor, central for the regulation of seedling photomorphogenesis. Here, we identified a B-BOX (BBX)-containing protein, BBX25/SALT TOLERANCE HOMOLOG, as an interacting partner of HY5, which has been previously found to physically interact with CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1). BBX25 physically interacts with HY5 both in vitro and in vivo. By physiological and genetic approaches, we showed that BBX25 is a negative regulator of seedling photomorphogenesis. BBX25 and its homolog BBX24 regulate deetiolation processes and hypocotyl shade avoidance response in an additive manner. Moreover, genetic relationships of bbx25 and bbx24 with hy5 and cop1 revealed that BBX25 and BBX24 additively enhance COP1 and suppress HY5 functions. BBX25 accumulates in a light-dependent manner and undergoes COP1-mediated degradation in dark and light conditions. Furthermore, a protoplast cotransfection assay showed that BBX24 and BBX25 repress BBX22 expression by interfering with HY5 transcriptional activity. As HY5 binds to the BBX22 promoter and promotes its expression, our results identify a direct mechanism through which the expression of BBX22 is regulated. We suggest that BBX25 and BBX24 function as transcriptional corepressors, probably by forming inactive heterodimers with HY5, downregulating BBX22 expression for the fine-tuning of light-mediated seedling development.

Function of B-BOX under shade.

Plants are capable of perceiving changes in the light environment and finely adjust their growth and development. Reductions of red to far-red ratio (R:FR) generated by an increase of the plant canopy above the plant are sensed by the phytochrome system triggering the shade-avoidance syndrome (SAS) that includes elongation of vegetative structures, reduction of branching and acceleration of flowering. Albeit the SAS is a strategy of major adaptative significance in plant communities, involving massive changes in gene expression, our knowledge of the SAS signaling network is still fragmented. By a selection and characterization of a T-DNA mutant with a long hypocotyl under shade, we identified BBX21, a protein with two B-box domains involved in the SAS. BBX21 belongs to a small eight member family of B-box containing proteins with both opposite and additive functions in the SAS signaling. BBX21 down-regulates the gene expression of auxin, brassinosteroid and ethylene signaling pathway components under shade. Furthermore BBX21 is a transcription factor that interacts genetically with COP1. We propose a model in which a dynamic balance of positive and negative B-box transcriptional regulators acts as a gas-and-brake mechanism into the COP1 signaling to regulate the expression of SAS.

AtBBX21 and COP1 genetically interact in the regulation of shade avoidance.

Plants grown at high densities perceive the reduction in the ratio of red (R) to far-red (FR) light as a warning of competition. This light signal triggers morphological responses such as hypocotyl and stem elongation, and acceleration of flowering, which are known collectively as the shade-avoidance syndrome (SAS). Mutations in the photomorphogenic repressor COP1 suppress the SAS, but how COP1 modulates these responses is uncertain. We identified a new mutant with altered responses to natural shade, named lhus (long hypocotyl under shade). lhus seedlings have longer hypocotyls than wild-type under a low R:FR ratio, but not under sunlight or darkness. The lhus phenotype is due to a mutation affecting a B-box zinc finger transcription factor encoded by At1g75540, a gene previously reported as AtBBX21 that interacts with COP1 to control de-etiolation. Mutations in genes encoding other members of this protein family also result in impaired SAS regulation. Under short-term canopy shade, LHUS/BBX21 acts as positive regulator of SAS genes such as PAR1, HFR1, PIL1 and ATHB2. In contrast, global expression analysis of wild-type and lhus/bbx21 seedlings revealed that a large number of genes involved in hormonal signalling pathways are negatively regulated by LHUS/BBX21 in response to long-term canopy shade, and this observation fits well with the phenotype of lhus/bbx21 seedlings grown under a low R:FR ratio. Moreover, the bbx21 bbx22 double mutation restored the SAS in the cop1 background. We propose that LHUS/BBX21 and other B-box-containing proteins, such as BBX22, act downstream of COP1, and play a central role in early and long-term adjustment of the SAS in natural environments.