Mutant selection in the self-incompatible plant radish (Raphanus sativus L. var. sativus) using two-step TILLING.

Radish (Raphanus sativus L. var. sativus), a widely cultivated root vegetable crop, possesses a large sink organ (the root), implying that photosynthetic activity in radish can be enhanced by altering both the source and sink capacity of the plant. However, since radish is a self-incompatible plant, improved mutation-breeding strategies are needed for this crop. TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful method used for reverse genetics. In this study, we developed a new TILLING strategy involving a two-step mutant selection process for mutagenized radish plants: the first selection is performed to identify a BC1M1 line, that is, progenies of M1 plants crossed with wild-type, and the second step is performed to identify BC1M1 individuals with mutations. We focused on Rubisco as a target, since Rubisco is the most abundant plant protein and a key photosynthetic enzyme. We found that the radish genome contains six RBCS genes and one pseudogene encoding small Rubisco subunits. We screened 955 EMS-induced BC1M1 lines using our newly developed TILLING strategy and obtained six mutant lines for the six RsRBCS genes, encoding proteins with four different types of amino acid substitutions. Finally, we selected a homozygous mutant and subjected it to physiological measurements.

Plant-plant interactions mediate the plastic and genotypic response of Plantago asiatica to CO2: an experiment with plant populations from naturally high CO2 areas.

BACKGROUND AND AIMS: The rising atmospheric CO2 concentration ([CO2]) is a ubiquitous selective force that may strongly impact species distribution and vegetation functioning. Plant-plant interactions could mediate the trajectory of vegetation responses to elevated [CO2], because some plants may benefit more from [CO2] elevation than others. The relative contribution of plastic (within the plant's lifetime) and genotypic (over several generations) responses to elevated [CO2] on plant performance was investigated and how these patterns are modified by plant-plant interactions was analysed. METHODS: Plantago asiatica seeds originating from natural CO2 springs and from ambient [CO2] sites were grown in mono stands of each one of the two origins as well as mixtures of both origins. In total, 1944 plants were grown in [CO2]-controlled walk-in climate rooms, under a [CO2] of 270, 450 and 750 ppm. A model was used for upscaling from leaf to whole-plant photosynthesis and for quantifying the influence of plastic and genotypic responses. KEY RESULTS: It was shown that changes in canopy photosynthesis, specific leaf area (SLA) and stomatal conductance in response to changes in growth [CO2] were mainly determined by plastic and not by genotypic responses. We further found that plants originating from high [CO2] habitats performed better in terms of whole-plant photosynthesis, biomass and leaf area, than those from ambient [CO2] habitats at elevated [CO2] only when both genotypes competed. Similarly, plants from ambient [CO2] habitats performed better at low [CO2], also only when both genotypes competed. No difference in performance was found in mono stands. CONCLUSION: The results indicate that natural selection under increasing [CO2] will be mainly driven by competitive interactions. This supports the notion that plant-plant interactions have an important influence on future vegetation functioning and species distribution. Furthermore, plant performance was mainly driven by plastic and not by genotypic responses to changes in atmospheric [CO2].

Foliage nitrogen turnover: differences among nitrogen absorbed at different times by Quercus serrata saplings.

BACKGROUND AND AIMS: Nitrogen turnover within plants has been intensively studied to better understand nitrogen use strategies. However, differences among the nitrogen absorbed at different times are not completely understood and the fate of nitrogen absorbed during winter is largely uncharacterized. In the present study, nitrogen absorbed at different times of the year (growing season, winter and previous growing season) was traced, and the within-leaf nitrogen turnover of a temperate deciduous oak Quercus serrata was investigated. METHODS: The contributions of nitrogen absorbed at the three different times to leaf construction, translocation during the growing season, and the leaf-level resorption efficiency during leaf senescence were compared using (15)N. KEY RESULTS: Winter- and previous growing season-absorbed nitrogen significantly contributed to leaf construction, although the contribution was smaller than that of growing season-absorbed nitrogen. On the other hand, the leaf-level resorption efficiency of winter- and previous growing season-absorbed nitrogen was higher than that of growing season-absorbed nitrogen, suggesting that older nitrogen is better retained in leaves than recently absorbed nitrogen. CONCLUSIONS: The results demonstrate that nitrogen turnover in leaves varies with nitrogen absorption times. These findings are important for understanding plant nitrogen use strategies and nitrogen cycles in forest ecosystems.

Allocation of nitrogen within the crown during leaf expansion in Quercus serrata saplings.

Early season leaf growth requires a large amount of nitrogen, and the amount of N provided for new leaf development has been well tested. Although shoot position within the crown strongly influences leaf properties, little is known about absorbed and remobilized nitrogen allocation in the tree crown. Thus, we investigated differences in the allocation of recently absorbed nitrogen in the tree crown. To quantify nitrogen allocation, we conducted 15N tracer experiments using potted saplings of the temperate deciduous oak (Quercus serrata Thunb. ex. Murray). Allocation of 15N within the crown varied significantly: the top leaves received more remobilized nitrogen than did the lateral leaves, suggesting that remobilized nitrogen is predominantly allocated to the top shoots, which are important for height growth. On the other hand, the proportion of currently-absorbed nitrogen to total nitrogen in the lateral leaves was more than twice that in the top leaves. We also detected the input and the output of nitrogen in the top leaves after the completion of leaf expansion, indicating that significant nitrogen cycling occurs even after full leaf expansion.

Gross nitrogen retranslocation within a canopy of Quercus serrata saplings.

Nitrogen (N) retranslocation within tree canopies has been intensively studied and assumed to function as a one-way process (e.g., from older to newer leaves). However, recent studies have found that both N output and input occur in individual leaves, suggesting that 'gross' N retranslocation exists behind 'net' N retranslocation. In the present study, the amount and direction of gross N retranslocation within a canopy of deciduous oak Quercus serrata Thunb. ex. Murray saplings were investigated. Labeling was conducted with leaves of Q. serrata saplings cultivated under conditions of low-N (LN) or high-N (HN) fertility. Subsequently, N movement within the canopy was traced. Leaves at two different positions in the canopy (top and lateral) were labeled to determine the direction of gross N retranslocation. To detect seasonal differences, the leaf-labeling experiment was conducted twice during the early and late phases of the growing season. In addition, to compare the quantitative importance of gross N retranslocation and root N uptake, the latter was determined by labeling Q. serrata roots. The N-labeling experiment revealed gross N retranslocation among leaves, i.e., from top to lateral, lateral to top and lateral to lateral positions. Gross N retranslocation was quantitatively more important than root uptake, especially for plants cultivated at LN fertility. Season also affected the amount of gross N retranslocation, and these effects differed between LN and HN fertilities. These findings suggest that N allocation within a canopy is controlled dynamically by both gross N output and input. The mechanisms controlling gross N output and input likely function as key determinants of N allocation within a tree canopy.