Interaction between sugar and abscisic acid signalling during early seedling development in Arabidopsis

Sugars regulate important processes and affect the expression of many genes in plants. Characterization of Arabidopsis (Arabidopsis thaliana) mutants with altered sugar sensitivity revealed the function of abscisic acid (ABA) signalling in sugar responses. However, the exact interaction between sugar signalling and ABA is obscure. Therefore ABA deficient plants with constitutive ABI4 expression (aba2-1/35S::ABI4) were generated. Enhanced ABI4 expression did not rescue the glucose insensitive (gin) phenotype of aba2 seedlings indicating that other ABA regulated factors are essential as well. Interestingly, both glucose and ABA treatment of Arabidopsis seeds trigger a post-germination seedling developmental arrest. The glucose-arrested seedlings had a drought tolerant phenotype and showed glucose-induced expression of ABSCISIC ACID INSENSITIVE3 (ABI3), ABI5 and LATE EMBRYOGENESIS ABUNDANT (LEA) genes reminiscent of ABA signalling during early seedling development. ABI3 is a key regulator of the ABA-induced arrest and it is shown here that ABI3 functions in glucose signalling as well. Multiple abi3 alleles have a glucose insensitive (gin) phenotype comparable to that of other known gin mutants. Importantly, glucose-regulated gene expression is disturbed in the abi3 background. Moreover, abi3 was insensitive to sugars during germination and showed sugar insensitive (sis) and sucrose uncoupled (sun) phenotypes. Mutant analysis further identified the ABA response pathway genes ENHANCED RESPONSE TO ABA1 (ERA1) and ABI2 as intermediates in glucose signalling. Hence, three previously unidentified sugar signalling genes have been identified, showing that ABA and glucose signalling overlap to a larger extend than originally thought. Bas J. W. Dekkers and Jolanda A. M. J. Schuurmans contributed equally to this paper.     

The ABI1 and ABI2 protein phosphatases 2C act in a negative feedback regulatory loop of the abscisic acid signalling pathway

The Arabidopsis ABI1 and ABI2 genes encode two protein serine/threonine phosphatases 2C (PP2C). These genes have been originally identified by the dominant mutations abi1--1 and abi2--1, which reduce the plant's responsiveness to the hormone abscisic acid (ABA). However, recessive mutants of ABI1 were recently shown to be supersensitive to ABA, which demonstrated that the ABI1 phosphatase is a negative regulator of ABA signalling. We report here the isolation and characterisation of the first reduction-of-function allele of ABI2, abi2--1R1. The in vitro phosphatase activity of the abi2--1R1 protein is approximately 100-fold lower than that of the wild-type ABI2 protein. Abi2--1R1 plants displayed a wild-type ABA sensitivity. However, doubly mutant plants combining the abi2--1R1 allele and a loss-of-function allele at the ABI1 locus were more responsive to ABA than each of the parental single mutants. These data indicate that the wild-type ABI2 phosphatase is a negative regulator of ABA signalling, and that the ABI1 and ABI2 phosphatases have overlapping roles in controlling ABA action. Measurements of PP2C activity in plant extracts showed that the phosphatase activity of ABI1 and ABI2 increases in response to ABA. These results suggest that ABI1 and ABI2 act in a negative feedback regulatory loop of the ABA signalling pathway.     

Analysis of natural allelic variation at seed dormancy loci of Arabidopsis thaliana

Arabidopsis accessions differ largely in their seed dormancy behavior. To understand the genetic basis of this intraspecific variation we analyzed two accessions: the laboratory strain Landsberg erecta (Ler) with low dormancy and the strong-dormancy accession Cape Verde Islands (Cvi). We used a quantitative trait loci (QTL) mapping approach to identify loci affecting the after-ripening requirement measured as the number of days of seed dry storage required to reach 50% germination. Thus, seven QTL were identified and named delay of germination (DOG) 1-7. To confirm and characterize these loci, we developed 12 near-isogenic lines carrying single and double Cvi introgression fragments in a Ler genetic background. The analysis of these lines for germination in water confirmed four QTL (DOG1, DOG2, DOG3, and DOG6) as showing large additive effects in Ler background. In addition, it was found that DOG1 and DOG3 genetically interact, the strong dormancy determined by DOG1-Cvi alleles depending on DOG3-Ler alleles. These genotypes were further characterized for seed dormancy/germination behavior in five other test conditions, including seed coat removal, gibberellins, and an abscisic acid biosynthesis inhibitor. The role of the Ler/Cvi allelic variation in affecting dormancy is discussed in the context of current knowledge of Arabidopsis germination.     

The Arabidopsis SUCROSE UNCOUPLED-6 gene is identical to ABSCISIC ACID INSENSITIVE-4: involvement of abscisic acid in sugar responses

In plants, sugars act as signalling molecules that control many aspects of metabolism and development. Arabidopsis plants homozygous for the recessive sucrose uncoupled-6 (sun6) mutation show a reduced sensitivity to sugars for processes such as photosynthesis, gene expression and germination. The sun6 mutant is insensitive to sugars that are substrates for hexokinase, suggesting that SUN6 might play a role in hexokinase-dependent sugar responses. The SUN6 gene was cloned by transposon tagging and analysis showed it to be identical to the previously described ABSCISIC ACID INSENSITIVE-4 (ABI4) gene. Our analysis suggests the involvement of abscisic acid and components of the abscisic acid signal transduction cascade in a hexokinase-dependent sugar response pathway. During the plant life cycle, SUN6/ABI4 may be involved in controlling metabolite availability in an abscisic acid- and sugar-dependent way.     

Sucrose-specific induction of anthocyanin biosynthesis in Arabidopsis requires the MYB75/PAP1 gene

Sugar-induced anthocyanin accumulation has been observed in many plant species. We observed that sucrose (Suc) is the most effective inducer of anthocyanin biosynthesis in Arabidopsis (Arabidopsis thaliana) seedlings. Other sugars and osmotic controls are either less effective or ineffective. Analysis of Suc-induced anthocyanin accumulation in 43 Arabidopsis accessions shows that considerable natural variation exists for this trait. The Cape Verde Islands (Cvi) accession essentially does not respond to Suc, whereas Landsberg erecta is an intermediate responder. The existing Landsberg erecta/Cvi recombinant inbred line population was used in a quantitative trait loci analysis for Suc-induced anthocyanin accumulation (SIAA). A total of four quantitative trait loci for SIAA were identified in this way. The locus with the largest contribution to the trait, SIAA1, was fine mapped and using a candidate gene approach, it was shown that the MYB75/PAP1 gene encodes SIAA1. Genetic complementation studies and analysis of a laboratory-generated knockout mutation in this gene confirmed this conclusion. Suc, in a concentration-dependent way, induces MYB75/PAP1 mRNA accumulation. Moreover, MYB75/PAP1 is essential for the Suc-mediated expression of the dihydroflavonol reductase gene. The SIAA1 locus in Cvi probably is a weak or loss-of-function MYB75/PAP1 allele. The C24 accession similarly shows a very weak response to Suc-induced anthocyanin accumulation encoded by the same locus. Sequence analysis showed that the Cvi and C24 accessions harbor mutations both inside and downstream of the DNA-binding domain of the MYB75/PAP1 protein, which most likely result in loss of activity.     

The Arabidopsis sugar-insensitive mutants sis4 and sis5 are defective in abscisic acid synthesis and response

Although soluble sugar levels affect many aspects of plant development and physiology, little is known about the mechanisms by which plants respond to sugar. Here we report the isolation of 13 sugar-insensitive (sis) mutants of Arabidopsis that, unlike wild-type plants, are able to form expanded cotyledons and true leaves when germinated on media containing high concentrations of glucose or sucrose. The sis4 and sis5 mutants are allelic to the ABA-biosynthesis mutant aba2 and the ABA-insensitive mutant abi4, respectively. In addition to being insensitive to glucose and sucrose, the sis4/aba2 and sis5/abi4 mutants also display decreased sensitivity to the inhibitory effects of mannose on early seedling development. Mutations in the ABI5 gene, but not mutations in the ABI1, ABI2 or ABI3 genes, also lead to weak glucose- and mannose-insensitive phenotypes. Wild-type and mutant plants show similar responses to the effects of exogenous sugar on chlorophyll and anthocyanin accumulation, indicating that the mutants are not defective in all sugar responses. These results indicate that defects in ABA metabolism and some, but not all, defects in ABA response can also alter response to exogenous sugar.     

Light-related loci controlling seed germination in Ler x Cvi and Bay-0 x Sha recombinant inbred-line populations of Arabidopsis thaliana

Background and Aims

Dormancy is a complex trait finely regulated by hormones and environmental factors. The phytochromes that sense red:far-red (R:FR) are the sole photoreceptors involved in the termination of dormancy and the induction of germination by light. The aims of this study were to identify and characterize loci controlling this process in seeds of Arabidopsis thaliana.

Methods

Recombinant inbred lines (RILs) derived from Landsberg erecta and Cape Verde Islands (Ler × Cvi), and Bayreuth and Shahdara (Bay-0 × Sha) were used to map loci related to light effects in seeds previously exposed to chilling and after-ripening periods.

Key Results

Substantial genetic variation was found between accessions of A. thaliana in the induction of germination by light. Twelve loci were identified under R, FR or darkness, some of which were novel loci: DOG8, DOG9, DOG13, DOG14 and DOG15 detected in the Ler × Cvi RIL population; and DOG10, DOG11 and DOG12 mapped in the Bay-0 × Sha RIL population. Furthermore, independent loci were mapped for the induction of germination by low fluence (DOG-LF1 and DOG-LF2) and very low fluence of light (DOG-VLF1) in the Ler × Cvi RIL population. Several loci were confirmed and characterized after different after-ripening and chilling treatments through near-isogenic lines (NILs) and heterogeneous inbred families (HIFs).

Conclusions

The results show that one group of loci act in a wide range of environmental scenarios, whereas a smaller group of loci are relevant only under a narrower set of conditions when the influence of the most-prevalent loci is reduced as a consequence of changes in the physiological status of the seeds. In addition, the identification of specific loci controlling the action modes of the phytochromes improves our understanding of the two independent signalling pathways that promote germination in response to light.Key words: Arabidopsis thaliana, dormancy, germination, phytochromes, very-low-fluence response (VLFR), low-fluence response (LFR), natural genetic variation, quantitative trait loci (QTL), recombinant inbred line (RIL), near-isogenic line (NIL) heterogeneous inbred family (HIF)     

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.     

Amino acid polymorphisms in strictly conserved domains of a P-type ATPase HMA5 are involved in the mechanism of copper tolerance variation in Arabidopsis

Copper (Cu) is an essential element in plant nutrition, but it inhibits the growth of roots at low concentrations. Accessions of Arabidopsis (Arabidopsis thaliana) vary in their tolerance to Cu. To understand the molecular mechanism of Cu tolerance in Arabidopsis, we performed quantitative trait locus (QTL) analysis and accession studies. One major QTL on chromosome 1 (QTL1) explained 52% of the phenotypic variation in Cu tolerance in roots in a Landsberg erecta/Cape Verde Islands (Ler/Cvi) recombinant inbred population. This QTL regulates Cu translocation capacity and involves a Cu-transporting P_1B-1-type ATPase, HMA5. The Cvi allele carries two amino acid substitutions in comparison with the Ler allele and is less functional than the Ler allele in Cu tolerance when judged by complementation assays using a T-DNA insertion mutant. Complementation assays of the ccc2 mutant of yeast using chimeric HMA5 proteins revealed that N923T of the Cvi allele, which was identified in the tightly conserved domain N(x)₆YN(x)₄P (where the former asparagine was substituted by threonine), is a cause of dysfunction of the Cvi HMA5 allele. Another dysfunctional HMA5 allele was identified in Chisdra-2, which showed Cu sensitivity and low capacity of Cu translocation from roots to shoots. A unique amino acid substitution of Chisdra-2 was identified in another strictly conserved domain, CPC(x)₆P, where the latter proline was replaced with leucine. These results indicate that a portion of the variation in Cu tolerance of Arabidopsis is regulated by the functional integrity of the Cu-translocating ATPase, HMA5, and in particular the amino acid sequence in several strictly conserved motifs.