Coordination of shoot photosynthetic carbon fixation with root inorganic nitrogen uptake optimizes plant performance in a fluctuating environment [1]. However, the molecular basis of this long-distance shoot-root coordination is little understood. Here we show that Arabidopsis ELONGATED HYPOCOTYL5 (HY5), a bZIP transcription factor that regulates growth in response to light [2, 3], is a shoot-to-root mobile signal that mediates light promotion of root growth and nitrate uptake. Shoot-derived HY5 auto-activates root HY5 and also promotes root nitrate uptake by activating NRT2.1, a gene encoding a high-affinity nitrate transporter [4]. In the shoot, HY5 promotes carbon assimilation and translocation, whereas in the root, HY5 activation of NRT2.1 expression and nitrate uptake is potentiated by increased carbon photoassimilate (sucrose) levels. We further show that HY5 function is fluence-rate modulated and enables homeostatic maintenance of carbon-nitrogen balance in different light environments. Thus, mobile HY5 coordinates light-responsive carbon and nitrogen metabolism, and hence shoot and root growth, in a whole-organismal response to ambient light fluctuations.

Phosphate (Pi) is a macronutrient that is essential for plant growth and development. However, the low mobility of Pi impedes uptake, thus reducing availability. Accordingly, plants have developed physiological strategies to cope with low Pi availability. Here, we report that the characteristic Arabidopsis thaliana Pi starvation responses are in part dependent on the activity of the nuclear growth-repressing DELLA proteins (DELLAs), core components of the gibberellin (GA)-signaling pathway. We first show that multiple shoot and root Pi starvation responses can be repressed by exogenous GA or by mutations conferring a substantial reduction in DELLA function. In contrast, mutants having enhanced DELLA function exhibit enhanced Pi starvation responses. We also show that Pi deficiency promotes the accumulation of a green fluorescent protein-tagged DELLA (GFP-RGA [repressor of ga1-3]) in root cell nuclei. In further experiments, we show that Pi starvation causes a decrease in the level of bioactive GA and associated changes in the levels of gene transcripts encoding enzymes of GA metabolism. Finally, we show that the GA-DELLA system regulates the increased root hair length that is characteristic of Pi starvation. In conclusion, our results indicate that DELLA-mediated signaling contributes to the anthocyanin accumulation and root architecture changes characteristic of Pi starvation responses, but do not regulate Pi starvation-induced changes in Pi uptake efficiency or the accumulation of selected Pi starvation-responsive gene transcripts. Pi starvation causes a reduction in bioactive GA level, which, in turn, causes DELLA accumulation, thus modulating several adaptively significant plant Pi starvation responses.

ORCID ID: 0000-0002-7224-8449 (Y.Z.).Mutations generated by CRISPR/Cas9 in Arabidopsis (Arabidopsis thaliana) are often somatic and are rarely heritable. Isolation of mutations in Cas9-free Arabidopsis plants can ensure the stable transmission of the identified mutations to next generations, but the process is laborious and inefficient. Here, we present a simple visual screen for Cas9-free T2 seeds, allowing us to quickly obtain Cas9-free Arabidopsis mutants in the T2 generation. To demonstrate this in principle, we targeted two sites in the AUXIN-BINDING PROTEIN1 (ABP1) gene, whose function as a membrane-associated auxin receptor has been challenged recently. We obtained many T1 plants with detectable mutations near the target sites, but only a small fraction of T1 plants yielded Cas9-free abp1 mutations in the T2 generation. Moreover, the mutations did not segregate in Mendelian fashion in the T2 generation. However, mutations identified in the Cas9-free T2 plants were stably transmitted to the T3 generation following Mendelian genetics. To further simplify the screening procedure, we simultaneously targeted two sites in ABP1 to generate large deletions, which can be easily identified by PCR. We successfully generated two abp1 alleles that contained 1,141-and 711-bp deletions in the ABP1 gene. All of the Cas9-free abp1 alleles we generated were stable and heritable. The method described here allows for effectively isolating Cas9-free heritable CRISPR mutants in Arabidopsis.

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Potassium (K+) and chloride (Cl-) are two essential elements for plant growth and development. While it is known that plants possess specific membrane transporters for transporting K+ and Cl-, it remains unclear if they actively use K+-coupled Cl- cotransporters (KCC), as used in animals, to transport K+ and Cl-. We have cloned an Oryza sativa cDNA encoding for a member of the cation-Cl- cotransporter (CCC) family. Phylogenetic analysis revealed that plant CCC proteins are highly conserved and that they have greater sequence similarity to the sub-family of animal K--Cl- cotransporters than to other cation-Cl- cotransporters. Real-time PCR revealed that the O. sativa cDNA, which was named OsCCC1, can be induced by KCl in the shoot and root and that the expression level was higher in the leaf and root tips than in any other part of the rice plant. The OsCCC1 protein was located not only in onion plasma membrane but also in O. sativa plasma membrane. The OsCCC1 gene-silenced plants grow more slowly than wild-type (WT) plants, especially under the KCl treatment regime. After 1 month of KCl treatment, the leaf tips of the gene-silenced lines were necrosed. In addition, seed germination, root length, and fresh and dry weight were distinctly lower in the gene-silenced lines than in WT plants, especially after KCl treatment. Analysis of Na+, K+, and Cl- contents of the gene-silenced lines and WT plants grown under the NaCl and KCl treatment regimes revealed that the former accumulated relatively less K+ and Cl- than the latter but that they did not differ in terms of Na+ contents, suggesting OsCCC1 may be involved in K+ and Cl- transport. Results from different tests indicated that the OsCCC1 plays a significant role in K+ and Cl- homeostasis and rice plant development.

The yeast (Schizosaccharomyces pombe) SOD2 (Sodium2) gene was introduced into Arabidopsis under the control of the cauliflower mosaic virus 35S promoter. Transformants were selected for their ability to grow on medium containing kanamycin. Southern-and northern-blot analyses confirmed that SOD2 was transferred into the Arabidopsis genome. There were no obvious morphological or developmental differences between the transgenic and wild-type (wt) plants. Several transgenic homozygous lines and wt plants (control) were evaluated for salt tolerance and gene expression. Overexpression of SOD2 in Arabidopsis improved seed germination and seedling salt tolerance. Analysis of Na ϩ and K ϩ contents of the symplast and apoplast in the parenchyma cells of the root cortex and mesophyll cells in the spongy tissue of the leaf showed that transgenic lines accumulated less Na ϩ and more K ϩ in the symplast than the wt plants did. The photosynthetic rate and the fresh weight of the transgenic lines were distinctly higher than that of wt plants after NaCl treatment. Results from different tests indicated that the expression of the SOD2 gene promoted a higher level of salt tolerance in vivo in transgenic Arabidopsis plants.A major factor impairing worldwide agricultural productivity is salinity, which is believed to affect nearly one-fifth of the world's irrigated land and resulted in a loss of 10 million ha of otherwise arable land each year (Boyer, 1982; Szaboles, 1987; Flowers and Yeo, 1995; Nelson et al., 1998). To solve the problems caused by salinity in agricultural areas, several approaches have been applied, such as irrigation with fresh water and improving soil drainage. However, these expensive solutions are not always practical. Therefore, the study of plant salt tolerance, with a view to identify and eventually to manipulate the genes involved in salt perception and responses, seems to be a promising approach (Zhu, 2000).Plant growth depends on mineral nutrients absorbed from the soil by roots. Although Na ϩ is a major cation present in the soil, it is not considered an essential mineral for most plants. In saline soils, high concentrations of Na ϩ disrupt the balance of other minerals such as K ϩ , thereby creating osmotic stress and causing secondary problems such as oxidative stress (Zhu, 2001). These adverse effects result in plant growth inhibition and even plant death.The mechanisms for plant cells to prevent excessive accumulation of Na ϩ in the cytosol are as follows.First, Na ϩ entry to plant cells may be restricted by selective ion uptake. However, nonselective cation channels have been proposed to mediate substantial Na ϩ entry into plant roots (Davenport and Tester, 2000; Demidchik and Tester, 2002). The cloned highaffinity potassium uptake transporter (HKT1) and low-affinity cation transporter (LCT1) affect Na ϩ permeability when expressed in yeast or oocytes, suggesting that they may also be considered Na ϩ influx transporters (Rubio et al., 1995; Schachtman et al., 1997; Liu et al., 2000).Second, inter...

Deer antlers are unique mammalian appendages in that each year they are cast and fully regenerate from permanent bony protuberances, called pedicles. In a previous study, we found that there is a difference in the degree of association between pedicle bone and its enveloping skin: tight at the distal third and loose at the proximal two thirds of a pedicle stump. The distal part has been termed the "potentiated" region, and the proximal part the "dormant" region. In the present study, pedicle stumps were artificially created in yearling sika deer by cutting off the tissue distal to either the potentiated or the dormant region. A piece of impermeable membrane was then inserted into the space between the bone and the skin of each treated pedicle stump, while the control pedicles had the same surgery without membrane insertion. The results showed that the inserted membrane blocked pedicle skin participation in the process of antler regeneration. All three potentiated bony pedicle stumps regenerated skin-less antlers; whereas, one of the three dormant bony pedicle stumps failed to regenerate any antler tissue. The other two dormant stumps eventually regenerated normal antlers; however, this only occurred after loss of the inserted membrane. No antler tissue regenerated from the dormant stumps while the inserted membrane remained in place (up to 55 days). All control pedicle stumps regenerated normal antlers. Therefore, we conclude that it is the pedicle bone, but not pedicle skin, that gives rise to regenerating antlers, and that pedicle bone can acquire the potential to regenerate an antler only when it is primed via interaction with its enveloping skin.

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