Javier F. Botto

Phytochrome A Mediates the Promotion of Seed Germination by Very Low Fluences of Light and Canopy Shade Light in Arabidopsis.

Seeds of the wild type (WT) and of the phyA and phyB mutants of Arabidopsis thaliana were exposed to single red light (R)/far-red light (FR) pulses predicted to establish a series of calculated phytochrome photoequilibria (Pfr/P). WT and phyB seeds showed biphasic responses to Pfr/P. The first phase, i.e. the very-low-fluence response (VLFR), occurred below Pfr/P = 10-1%. The second phase, i.e. the low-fluence response, occurred above Pfr/P = 3%. The VLFR was similarly induced by either a FR pulse saturating photoconversion or a subsaturating R pulse predicted to establish the same Pfr/P. The VLFR was absent in phyA seeds, which showed a strong low-fluence response. In the field, even brief exposures to the very low fluences of canopy shade light (R/FR ratio < 0.05) promoted germination above dark controls in WT and phyB seeds but not in the phyA mutant. Seeds of the phyA mutant germinated normally under canopies providing higher R/FR ratios or under deep canopy shade light supplemented with R from light-emitting diodes. We propose that phytochrome A mediates VLFR of A. thaliana seeds.

The BBX family of plant transcription factors

The B-box (BBX) proteins are a class of zinc-finger transcription factors containing a B-box domain with one or two B-box motifs, and sometimes also feature a CCT (CONSTANS, CO-like, and TOC1) domain. BBX proteins are key factors in regulatory networks controlling growth and developmental processes that include seedling photomorphogenesis, photoperiodic regulation of flowering, shade avoidance, and responses to biotic and abiotic stresses. In this review we discuss the functions of BBX proteins and the role of B-box motif in mediating transcriptional regulation and protein-protein interaction in plant signaling. In addition, we provide novel insights into the molecular mechanisms of their action and the evolutionary significance of their functional divergence.

The Arabidopsis B-BOX Protein BBX25 Interacts with HY5, Negatively Regulating BBX22 Expression to Suppress Seedling Photomorphogenesis

The B-box domains of BBX25 physically interact with the bZIP domain of HY5, and BBX24 and BBX25 inhibit the activation of BBX22 expression by HY5, probably by forming inactive heterodimers. In contrast with their role during deetiolation, BBX24 and BBX25 can switch roles and function independently of HY5 in the hypocotyl shade avoidance response. 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.

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.

Role of phytochrome B in the induction of seed germination by light in Arabidopsis thaliana

Summary The role played by phytochrome B in seed germination responses to light pulses was investigated by comparing wild-type (WT) and phyB-1 -mutant seeds of Arabidopsis thaliana (L.) Heynh. Similar dark germination rates were observed in WT and phyB-1 seeds stored dry for at least one month. The response of WT seeds to light pulses that established a series of calculated phytochrome photoequilibria was biphasic. In the first phase, i.e. the very-low-fluence response, similar slopes were observed in WT and phyB-1 -mutant seeds. In the second phase, i.e. the low-fluence response, the slope of the WT was 30 to 930% higher (depending on the batch, duration of dry storage, and temperature pretratements) than the slope of the phyB-1 mutant. Both hourly pulses of FR and continuous FR promoted germination above dark controls in both genotypes. Phytochrome B is proposed to make a higher contribution to low-fluence than very-low-fluence germination responses of Arabidopsis thaliana seeds.

Germination variation in Arabidopsis thaliana accessions under moderate osmotic and salt stresses

BACKGROUND Water and salt stresses are two important environmental factors that limit the germination of seeds in most ecological environments. Most studies conducted so far to address the genetic basis of the above phenomenon have used stress conditions that are much more extreme than those found in natural environments. Furthermore, although an excess of ions and water restrictions have similar osmotic effects on germination, the common and divergent signalling components mediating the effects of both factors remain unknown. METHODS The germination of seeds was compared under solutions of NaCl (50 mm) and polyethylene glycol (PEG, -0·6 MPa), that establish mild stress conditions, in 28 Arabidopsis thaliana accessions. Because Bayreuth (Bay) and Shadara (Sha) accessions showed contrasting sensitivity responses to both stresses, a quantitative trait locus (QTL) analysis was carried out using Bay × Sha recombinant inbred lines (RILs) to identify loci involved in the control of germination under mild salt and osmotic stresses. KEY RESULTS Two loci associated with the salt sensitivity response, named SSR1 and SSR2 QTLs, and four loci for the osmotic sensitivity response, named OSR1-OSR4 QTLs, were mapped. The effects of the SSR1 QTL on toxic salt sensitivity, and the osmotic contribution of OSR1, were confirmed by heterogeneous inbred families (HIFs). Whilst the SSR1 QTL had a significant effect under a wide range of NaCl concentrations, the OSR1 QTL was confirmed only under moderate drought stress. Interestingly the OSR1 QTL also showed pleiotropic effects on biomass accumulation in response to water deficit. CONCLUSIONS The regulation of germination under moderate salt and osmotic stresses involves the action of independent major loci, revealing the existence of loci specifically associated with the toxic component of salt and not just its osmotic effect. Furthermore, this work demonstrates that novel loci control germination under osmotic stress conditions simulating more realistic ecological environments as found by populations of seeds in nature.

Phytochrome B increases drought tolerance by enhancing ABA sensitivity in Arabidopsis thaliana

Phytochrome B (phyB) can adjust morphological and physiological responses according to changes in the red:far-red (R:FR) ratio. phyB-driven acclimation of plants to open environments (high R:FR ratio) increases carbon gain at the expense of increased water loss. This behaviour alleviates stressful conditions generated by an excess of light, but increases the chances of desiccation. Here we evaluated how phyB modulates this drought-tolerance response by comparing wild-type Arabidopsis thaliana adult plants to the null phyB in response to water shortage. phyB wilted before the wild type, and this was due to phyB maintaining open stomata under a reduction in soil water availability. Although phyB presented enhanced ABA levels under well-watered conditions, this mutant was less sensitive than the wild type in diminishing stomatal conductance in response to exogenous ABA application. Reduced sensitivity to ABA in phyB correlated with a lower expression of ABCG22, which encodes a putative ABA influx transporter, and PYL5, which encodes a soluble ABA receptor. Furthermore, the expression of RAB18 and RD29A, both typical ABA-induced genes, was lower in phyB than the wild type after ABA treatment. We propose that phyB contributes to the acclimation of plants to open environments by enhancing ABA sensitivity when soil water becomes limiting.

The Cape Verde Islands Allele of Cryptochrome 2 Enhances Cotyledon Unfolding in the Absence of Blue Light in Arabidopsis

We analyzed the natural genetic variation between Landsburg erecta (Ler) and Cape Verde Islands (Cvi) accessions by studying 105 recombinant inbred lines to search for players in the regulation of sensitivity to light signals perceived by phytochromes in etiolated seedlings of Arabidopsis. In seedlings grown under hourly pulses of far-red (FR) light, we identified three quantitative trait loci (QTLs; VLF3, VLF4, and VLF5) for hypocotyl growth inhibition and three different QTLs (VLF6, VLF7, and VLF1) for cotyledon unfolding. This indicates that different physiological outputs have selective regulation of sensitivity during de-etiolation. Ler alleles, compared with Cvi alleles, of VLF3, VLF4, VLF5, VLF7, and VLF1 enhanced, whereas the Ler allele of VLF6 reduced, the response to pulses of FR. We confirmed and narrowed down the position of some QTLs by using near-isogenic lines. VLF6 mapped close to the CRY2 (cryptochrome 2) gene. Transgenic Ler seedlings expressing the Cvi allele of CRY2 showed enhanced cotyledon unfolding under hourly pulses of FR compared with the wild type or transgenics expressing the CRY2-Ler allele. This response required phytochrome A. The cry1 cry2 double mutant lacking both cryptochromes showed reduced cotyledon unfolding under FR pulses. Because the CRY2-Cvi is a gain-of-function allele compared with CRY2-Ler, cryptochrome activity correlates positively with cotyledon unfolding under FR pulses. We conclude that the blue light photoreceptor cryptochrome 2 can modulate seedling photomorphogenesis in the absence of blue light. In addition to the nuclear loci, we identified cytoplasmic effects on seedling de-etiolation.

Genetic mapping of natural variation in a shade avoidance response: ELF3 is the candidate gene for a QTL in hypocotyl growth regulation

When plants become shaded by neighbouring plants, they perceive a decrease in the red/far-red (R/FR) ratio of the light environment, which provides an early and unambiguous warning of the presence of competing vegetation. The mechanistic bases of the natural genetic variation in response to shade signals remain largely unknown. This study demonstrates that a wide range of genetic variation for hypocotyl elongation in response to an FR pulse at the end of day (EOD), a light signal that simulates natural shade, exists between Arabidopsis accessions. A quantitative trait locus (QTL) mapping analysis was done in the Bayreuth×Shahdara recombinant inbred line population. EODINDEX1 is the most significant QTL identified in response to EOD. The Shahdara alleles at EODINDEX1 caused a reduced response to shade as a consequence of an impaired hypocotyl inhibition under white light, and an accelerated leaf movement rhythm, which correlated positively with the pattern of circadian expression of clock genes such as PRR7 and PRR9. Genetic and quantitative complementation analyses demonstrated that ELF3 is the most likely candidate gene underlying natural variation at EODINDEX1. In conclusion, ELF3 is proposed as a component of the shade avoidance signalling pathway responsible for the phenotypic differences between Arabidopsis populations in relation to adaptation in a changing light environment.

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.