Joanna Putterill

Distinct Roles of GIGANTEA in Promoting Flowering and Regulating Circadian Rhythms in Arabidopsis

The circadian clock acts as the timekeeping mechanism in photoperiodism. In Arabidopsis thaliana, a circadian clock–controlled flowering pathway comprising the genes GIGANTEA (GI), CONSTANS (CO), and FLOWERING LOCUS T (FT) promotes flowering specifically under long days. Within this pathway, GI regulates circadian rhythms and flowering and acts earlier in the hierarchy than CO and FT, suggesting that GI might regulate flowering indirectly by affecting the control of circadian rhythms. We studied the relationship between the roles of GI in flowering and the circadian clock using late elongated hypocotyl circadian clock associated1 double mutants, which are impaired in circadian clock function, plants overexpressing GI (35S:GI), and gi mutants. These experiments demonstrated that GI acts between the circadian oscillator and CO to promote flowering by increasing CO and FT mRNA abundance. In addition, circadian rhythms in expression of genes that do not control flowering are altered in 35S:GI and gi mutant plants under continuous light and continuous darkness, and the phase of expression of these genes is changed under diurnal cycles. Therefore, GI plays a general role in controlling circadian rhythms, and this is different from its effect on the amplitude of expression of CO and FT. Functional GI:green fluorescent protein is localized to the nucleus in transgenic Arabidopsis plants, supporting the idea that GI regulates flowering in the nucleus. We propose that the effect of GI on flowering is not an indirect effect of its role in circadian clock regulation, but rather that GI also acts in the nucleus to more directly promote the expression of flowering-time genes.

The Molecular Basis of Temperature Compensation in the Arabidopsis Circadian Clock

Circadian clocks maintain robust and accurate timing over a broad range of physiological temperatures, a characteristic termed temperature compensation. In Arabidopsis thaliana, ambient temperature affects the rhythmic accumulation of transcripts encoding the clock components TIMING OF CAB EXPRESSION1 (TOC1), GIGANTEA (GI), and the partially redundant genes CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY). The amplitude and peak levels increase for TOC1 and GI RNA rhythms as the temperature increases (from 17 to 27°C), whereas they decrease for LHY. However, as temperatures decrease (from 17 to 12°C), CCA1 and LHY RNA rhythms increase in amplitude and peak expression level. At 27°C, a dynamic balance between GI and LHY allows temperature compensation in wild-type plants, but circadian function is impaired in lhy and gi mutant plants. However, at 12°C, CCA1 has more effect on the buffering mechanism than LHY, as the cca1 and gi mutations impair circadian rhythms more than lhy at the lower temperature. At 17°C, GI is apparently dispensable for free-running circadian rhythms, although partial GI function can affect circadian period. Numerical simulations using the interlocking-loop model show that balancing LHY/CCA1 function against GI and other evening-expressed genes can largely account for temperature compensation in wild-type plants and the temperature-specific phenotypes of gi mutants.

Molecular genetics of gynoecium development in Arabidopsis.

Carpels are the ovule-bearing structural units in angiosperms. In Arabidopsis, the specification of carpel identity is achieved by at least two separate pathways: a pathway mediated by the C class gene AG and an AG-independent pathway. Both pathways are negatively regulated by A class genes. Two genes, SPT and CRC, can promote differentiation of carpel tissue independently of AG and are thus components of the AG-independent pathway. CRC and SPT appear to act in a redundant manner to promote the differentiation of subsets of carpel tissues. The carpel primordium is subdivided into regional domains, both medial versus lateral and abaxial versus adaxial. Based on morphological and gene expression analyses, it appears likely that these domains define developmental compartments. The medial domain appears fated to differentiate into the marginal tissue types of the carpel (septum with transmitting tract and placenta with ovules), whereas the lateral domain gives rise to the ovary walls. The expression of ETT defines the abaxial domain, and this gene is involved in the abaxial-adaxial and, possibly, the apical-basal patterning of tissues in the carpel. Once regional domains have been established, the differentiation of tissue and cell types occurs. The MADS-box gene FUL and AGLI/5 are involved in the differentiation of specific tissue types in the valves and valve margins. Thus, the genes identified can be arranged in a functional hierarchy: specification of carpel identity, patterning of the carpel primordium and directing the differentiation of the specialized tissues of the carpel.

Arabidopsis GIGANTEA protein is post-transcriptionally regulated by light and dark.

GIGANTEA (GI) is a key regulator of photoperiodic flowering in Arabidopsis and encodes a protein with no domains of known biochemical function. Expression of GI mRNA is controlled by the circadian clock, but GI protein accumulation has not been previously investigated. We generated plants that produced functional epitope‐tagged GI to enable us to track the protein through the daily cycle. Here we show that GI protein levels oscillate when either constitutively overexpressed or driven by its promoter and that its accumulation is modulated by day length as well as by phase‐specific factors. Also, we demonstrate that one of the mechanisms underlying GI protein oscillation occurs post‐translationally via dark‐induced proteolysis by the 26S proteasome.

The Medicago FLOWERING LOCUS T Homolog, MtFTa1, Is a Key Regulator of Flowering Time

FLOWERING LOCUS T (FT) genes encode proteins that function as the mobile floral signal, florigen. In this study, we characterized five FT-like genes from the model legume, Medicago (Medicago truncatula). The different FT genes showed distinct patterns of expression and responses to environmental cues. Three of the FT genes (MtFTa1, MtFTb1, and MtFTc) were able to complement the Arabidopsis (Arabidopsis thaliana) ft-1 mutant, suggesting that they are capable of functioning as florigen. MtFTa1 is the only one of the FT genes that is up-regulated by both long days (LDs) and vernalization, conditions that promote Medicago flowering, and transgenic Medicago plants overexpressing the MtFTa1 gene flowered very rapidly. The key role MtFTa1 plays in regulating flowering was demonstrated by the identification of fta1 mutants that flowered significantly later in all conditions examined. fta1 mutants do not respond to vernalization but are still responsive to LDs, indicating that the induction of flowering by prolonged cold acts solely through MtFTa1, whereas photoperiodic induction of flowering involves other genes, possibly MtFTb1, which is only expressed in leaves under LD conditions and therefore might contribute to the photoperiodic regulation of flowering. The role of the MtFTc gene is unclear, as the ftc mutants did not have any obvious flowering-time or other phenotypes. Overall, this work reveals the diversity of the regulation and function of the Medicago FT family.

The Carboxylesterase Gene Family from Arabidopsis thaliana

Carboxylesterases hydrolyze esters of short-chain fatty acids and have roles in animals ranging from signal transduction to xenobiotic detoxification. In plants, however, little is known of their roles. We have systematically mined the genome from the model plant Arabidopsis thaliana for carboxylesterase genes and studied their distribution in the genome and expression profile across a range of tissues. Twenty carboxylesterase genes (AtCXE) were identified. The AtCXE family shares conserved sequence motifs and secondary structure characteristics with carboxylesterases and other members of the larger α/β hydrolase fold superfamily of enzymes. Phylogenetic analysis of the AtCXE genes together with other plant carboxylesterases distinguishes seven distinct clades, with an Arabidopsis thaliana gene represented in six of the seven clades. The AtCXE genes are widely distributed across the genome (present in four of five chromosomes), with the exception of three clusters of tandemly duplicated genes. Of the interchromosomal duplication events, two have been mediated through newly identified partial chromosomal duplication events that also include other genes surrounding the AtCXE loci. Eighteen of the 20 AtCXE genes are expressed over a broad range of tissues, while the remaining 2 (unrelated) genes are expressed only in the flowers and siliques. Finally, hypotheses for the functional roles of the AtCXE family members are presented based on the phylogenetic relationships with other plant carboxylesterases of known function, their expression profile, and knowledge of likely esterase substrates found in plants.

NODULE ROOT and COCHLEATA Maintain Nodule Development and Are Legume Orthologs of Arabidopsis BLADE-ON-PETIOLE Genes

Medicago truncatula NOOT and Pisum sativum COCH were found to maintain nodule identity during symbiotic interactions with rhizobia and were identified as orthologs of Arabidopsis BLADE-ON-PETIOLE genes, which are involved in leaf and flower development. During their symbiotic interaction with rhizobia, legume plants develop symbiosis-specific organs on their roots, called nodules, that house nitrogen-fixing bacteria. The molecular mechanisms governing the identity and maintenance of these organs are unknown. Using Medicago truncatula nodule root (noot) mutants and pea (Pisum sativum) cochleata (coch) mutants, which are characterized by the abnormal development of roots from the nodule, we identified the NOOT and COCH genes as being necessary for the robust maintenance of nodule identity throughout the nodule developmental program. NOOT and COCH are Arabidopsis thaliana BLADE-ON-PETIOLE orthologs, and we have shown that their functions in leaf and flower development are conserved in M. truncatula and pea. The identification of these two genes defines a clade in the BTB/POZ-ankyrin domain proteins that shares conserved functions in eudicot organ development and suggests that NOOT and COCH were recruited to repress root identity in the legume symbiotic organ.

The Expression of Petunia Strigolactone Pathway Genes is Altered as Part of the Endogenous Developmental Program

Analysis of mutants with increased branching has revealed the strigolactone synthesis/perception pathway which regulates branching in plants. However, whether variation in this well conserved developmental signaling system contributes to the unique plant architectures of different species is yet to be determined. We examined petunia orthologs of the Arabidopsis MAX1 and MAX2 genes to characterize their role in petunia architecture. A single ortholog of MAX1, PhMAX1 which encodes a cytochrome P450, was identified and was able to complement the max1 mutant of Arabidopsis. Petunia has two copies of the MAX2 gene, PhMAX2A and PhMAX2B which encode F-Box proteins. Differences in the transcript levels of these two MAX2-like genes suggest diverging functions. Unlike PhMAX2B, PhMAX2A mRNA levels change in leaves of differing age/position on the plant. Nonetheless, this gene functionally complements the Arabidopsis max2 mutant indicating that the biochemical activity of the PhMAX2A protein is not significantly different from MAX2. The expression of the petunia strigolactone pathway genes (PhCCD7, PhCCD8, PhMAX1, PhMAX2A, and PhMAX2B) was then further investigated throughout the development of wild-type petunia plants. Three of these genes showed changes in mRNA levels over a development series. Alterations to the expression patterns of these genes may influence the branching growth habit of plants by changing strigolactone production and/or sensitivity. These changes could allow both subtle and dramatic changes to branching within and between species.

Proteins with the potential to protect plants from Al3+ toxicity

The acidic polypeptides, poly(l-aspartic acid) and poly(l-glutamic acid) and the serum protein, transferrin, have been shown to bind Al3+ in a number of biochemical and functional assays. These compounds were able to relieve the inhibitory effect of Al3+ upon the activity of cAMP phosphodiesterase stimulated by Ca2+-calmodulin. In addition, the Al3+-induced structural changes in calmodulin, as measured by 8-anilo-1-naphthalenesulphonic acid fluorescence, were reduced by these compounds. Finally, equilibrium dialysis has confirmed substantial binding of Al3+ by the acidic polypeptides and transferrin. Citric acid, a significant chelator of Al3+, also relieved the effects of Al3+ upon the activity of phosphodiesterase and the structure of calmodulin. However, proteins from the central nervous system, enkephalins and metallothionein (a heavy metal-binding protein) were ineffective.

MDH1: an apple homeobox gene belonging to the BEL1 family.

Differential display was used to isolate genes differentially expressed early in fruit development of apple (Malus domestica Borkh.). This approach resulted in the isolation of MDH1, a homeobox gene with a homeodomain similar to that of BELL1 (BEL1), which is involved in regulation of ovule development in Arabidopsis. However, outside the homeodomain MDH1 is quite different from BEL1. In apple, MDH1 mRNA was predominantly found in flowers, expanding leaves and expanding fruit. In pre-anthesis flowers, in situ hybridization showed that MDH1 mRNA accumulated in ovules. To further investigate the function of this new homeobox gene, MDH1 was transformed into Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter. The transgenic Arabidopsis plants showed dwarfing, reduced fertility and changes in carpel and fruit (silique) shape. The size and shape of the cells in the transgenic fruit was irregular. Both the transgenic phenotypes in Arabidopsis and the expression pattern of this gene in apple are consistent with the idea that MDH1 is likely to play an important role in control of plant fertility.