Our results show that the greatest variation in stem water d18O and plant water sources occurred during the frontal season and initiation of the dry season in February, whenG. floribundum was shedding old leaves and growing new leaves, but P. piscipula maintained its leaves from the previous wet season . Contrary to what was expected, P. piscipula took water primarily from shallow sources regardless of the month, although some contribution from deeper sources has the potential to occur. Rain also appeared to be an important source for this species. This implies that P. piscipula could have a very well developed shallow root system that allows rapid water uptake after a precipitation event. On the other hand, G. floribundum took water from topsoil and bedrock, the latter being a more important source in the dry season. This suggests a deeper root system than G. floribundum. Overall, our results indicate that the contrasting early and late dry season leaf loss phenology of these two species is not simply determined by rooting depth, but rather a more complicated suite of species-based characteristics based on opportunistic use of dynamic water sources, the balance between carbon gain and water loss, and maintenance of water potential at the end of the dry season. These results are consistent with other studies demonstrating a broad array of coordinated strategies for dealing with seasonal drought in tropical forests . A primary factor determining differences in leaf loss phenology between the two studied species appears to be the maintenance of water potential. G. floribundum consistently exhibited more positive water potential values than P. piscipula, suggesting that G. floribundum has a limited capacity to tolerate negative water potential and moderates water use in a manner that maintains bulk leaf water potential at relatively more positive values compared to P. piscipula . This could provide an advantage of maximizing carbon gain during the dry season when light availability is high.
Leaf organic d13C and d18O values support this observation, because P. piscipula showed consistently higher d13C values than G. floribundum ,vertical farm coupled with lower d18O indicating that the decrease in photosynthetic carbon isotope discrimination was associated with greater stomatal conductance and greater photosynthesis . Greater photosynthesis in P. piscipula is consistent with maintaining a canopy of leaves later into the dry season. Thus, our results are most consistent with maintenance plant water potential to maximize carbon gain during the onset of the dry season. The observation that P. piscipula appeared to use shallower water sources and maintained its canopy of leaveslater into the dry season was not expected. Part of this pattern is driven by the capability of P. piscipula to utilize dynamic sources of water, such as the cold front precipitation during the frontal season . This makes sense, because the Laja bedrock layer was a poor source of water at all times, and soil pockets, which are available, but heterogeneous in distribution, were always better sources of water than rock layers . Water content of soil/ bedrock sources changed along the year suggesting a different seasonal contribution to plant water uptake. The hg of topsoil in the wet and frontal seasons were very similar and three times greater than values measured in the dry season. The Sascab bedrock layer could be a significant source of water in the wet and frontal seasons, but not in the dry season. Soil pockets had two times more water than topsoil in the dry season suggesting that they could be an important source of water for trees during the dry season. In the dry season, the rock profile had hg between 1 and 5 % but nearly all were less than 1 %. These values were slightly lower than those reported by Querejeta et al. and Hasselquist et al. in nearby areas, which suggest that the bedrock was subjected to a greater evaporation during this study. The d18O values of water in this study integrated processes ranging from evaporation of soil and bedrock water sources, transpiration of tree species, and precipitation events. In the wet season, enriched values of d18O of water in topsoil 10–15 cm and trees revealed the occurrence of a depleted precipitation event that occurred on October 21, 1 day before sampling, bringing 19.7 mm of water . Furthermore, a frontal system including cold front #3, the tropical wave #37, as well as the remnants of tropical storm Kiko that formed in the pacific, converged on the study area days before the wet season sampling in October 2007 .
Hurricanes, tropical storms and cold fronts generally have lower stable isotope ratios than convective precipitation events . For example, Perry et al. recorded d18O values of -9.91 % for precipitation during tropical storm Mitch in 1998, and precipitation events ranging from -6 to -10 % for d18O have been recorded in the vicinities of the study area . Consequently, depleted oxygen values in soil 15–30 cm and P. piscipula and G. floribundum trees could be accredited to precipitation originated from these events. Soil pockets also showed more negative values than rock, suggesting that depleted rain water reached this layer. During dry season measurements in February 2008, the d18O of topsoil 0–15 cm was more positive than groundwater suggesting another depleted source of water. Cold front #29, which occurred 4 days before sampling and brought 33.9 mm , could be the main source of water. The strength of the dry season promoted the enrichment of all water sources respect to earlier samplings. However, the more negative value of topsoil from 0 to 5 cm could be affected by dew water since this soil sampling was done early in the morning. More negative d18O values in topsoil than ground water have also been observed by Saha et al. in similar environmental conditions in Miami, associated with water condensation occurring at night in the upper soil layers. Condensation has been shown to deplete d18O soil water 10–15 cm depth by 5 % . Condensation can also account for up to 47 % of total transpiration . Surface dew is easily generated when temperatures go below the dew point at night or in early morning . Under tropical conditions in Tahiti, Clus et al. reported average dew yields of 0.102 mm of dew during the dry season. Therefore, condensed water could be an important source for P. piscipula. Overall, our results indicate that variation in phenology between these two deciduous tropical dry-forest tree species, which vary in the timing of their deciduousness, is not akin to the relatively large variation in rooting depth that can occur between tropical evergreen and deciduous species , but rather reflects the diversity of plant physiological strategies that occur in tropical forest .Insertional mutagenesis is a powerful strategy for gene identification and functional genomics in plants . While the T-DNA approach is applicable to the model plants Arabidopsis and rice, where effective transformation methods are available,nft vertical farming it may not be feasible in many other plant species whose transformation is inefficient.
Transposon can be alternatively used for insertional mutagenesis in those plants, since the generation of new insertions occurs through crossing or propagation rather than through transformation. Supported by the United States National Science Foundation-Plant Genome Program . S.Q. was supported by the Research Start-up Grants of Zhejiang Academy of Agricultural Sciences , China. P.B.F.O. was supported by EU FP5 and FP6 projects CerealGene Tags and CEDROME . J.-S. J. was supported by the World Class University and the Crop Functional Genomics Center projects, Korean Ministry of Education, Science and Technology, Korea.The maize Ac-Ds transposable element has been shown to be active in the plant kingdom widely .Ds insertion libraries have been generated in Arabidopsisand rice . However, the current strategies of transposon tagging are usually slow and labor intensive and have several drawbacks. For example, in the presence of Ac transposase , transposed Ds elements may continue secondary transpositions. Unstable Ds insertions and serial transposition events may cause untagged mutations because imprecise excision or a transposition footprint can result in a mutation that is no longer associated with the transposon . Another problem is that the Ac-Ds transposable elements are highly active in rice and can transpose early in newly transformed callus cells , which results in many sibling plants carrying the same Ds insertions and consequently decreasing gene tagging efficiency. In the present study, we constructed 12 Ac-Ds transposon tagging vectors based on three approaches: AcTPase controlled by glucocorticoid binding domain/VP16 acidic activation domain/Gal4 DNA-binding domain chemical inducible expression system; deletion of AcTPase via Crelox site-specific recombination that was initially triggered by Ds excision; and suppression of early transposition events in transformed rice callus through a dual-functional hygromycin resistance gene in a novel Ds element. We have tested these vectors in transgenic rice and characterized the transposition events. Our results showed that these vectors are useful in functional genomics of rice and they will be useful for other crop plants as well.We constructed Ac-Ds transposon tagging vectors using a GVG-inducible expression system . The vectors pJJ86 and pDs-Ac-GVG carry an in cis two-element system that consists of Ds, 35S:GVG that expresses the chimeric GVG transcription activator, and AcTPase controlled by a GVG-inducible promoter. The inducible promoter is transactivated through interaction between GVG and the 4xGAL4-upstream activating sequence .
The transactivating activity of GVG is regulated by treatment with the steroid chemical dexamethasone . The Ds element in pJJ86 contains the 4x CaMV 35S enhancers for activation tagging , while the Ds in pDsAc-GVG does not. Excision of Ds from pJJ86 can be detected because in the resulting T-DNA fragment, the β-glucuronidase gene is driven by a CaMV 35S promoter. We also constructed a two-vector tagging system in which GVG-inducible AcTPase and Ds are in separate vectors . The strategy of the two-vector system is that transgenic plants carrying the GVG-inducible AcTPase and Ds are generated, respectively, and the AcTPase and Ds are combined in F1 by genetic crosses. In this case, Ds is mobilized in the presence of AcTPase in F1 plants, but stabilized after it is uncoupled from AcTPase in the subsequent generation. To test whether the inducible Ac-Ds system is functional in rice, we transformed rice cultivar Nipponbare with pJJ86. Independently transformed rice calli were cultured for 5 d on media with DEX to induce expression of AcTPase. Because Ds transposition can be detected by GUS activity, the DEX-treated calli and untreated controls were stained for GUS activity. DEX treatment of pJJ86-transformed calli exhibited stronger GUS staining than controls , indicating that the DEXinducible system in this vector is functional in rice. At the same time, there was low background of GUS activity in the untreated rice calli , suggesting that some background transposition occurred in the pJJ86 transformants.Because the Ac-Ds transposable elements are active in newly transformed callus cells and early transposition events lead to the same Ds insertions in sibling plants, we constructed a novel Ds element, designated HPT-Ds, and used the hygromycin resistance gene to suppress transposition. The pHPT-Ds1 vector carrying HPT-Ds and GVGinducible AcTPase in cis is shown in Figure 1E. The HPT gene in HPT-Ds has the same intron and triple splice acceptors as in the gene-trap Ds . Because HPT-Ds is immediately downstream of maize ubiquitin 1 promoter in T-DNA, the Ubi:HPT-Ds fusion confers hygromycin resistance, and transformed rice cells are thereby selected on hygromycin media. In case of transposition, HPT-Ds in the rice genome may not have a promoter nearby for transcription and the rice cells lose hygromycin resistance and can be counter-selected by hygromycin. To examine the efficacy of the HPT-Ds element, we made a test construct containing Ubi:HPT-Ds and confirmed the function of the Ubi-driving HPT gene in a rice transformation experiment. A total of 250 rice calli were transformed using a particle bombardment method and hygromycin-resistant cells were selected from 30 callus explants after 50 d of selection on hgromycin media. In constructing the pHPT-Ds1 vector, HPT-Ds was cloned between Ubi and GUS so that transposant cells canbe detected by GUS assay. The pHPT-Ds2 vector is similar to pHPT-Ds1 except that pHPT-Ds2 carries a Bar gene and transposition can be selected by herbicide resistance . pHPT-Ds1 was introduced into rice cultivar Nipponbare.