A defensin-like protein drives cadmium efflux and allocation in rice.

Pollution by heavy metals limits the area of land available for cultivation of food crops. A potential solution to this problem might lie in the molecular breeding of food crops for phytoremediation that accumulate toxic metals in straw while producing safe and nutritious grains. Here, we identify a rice quantitative trait locus we name cadmium (Cd) accumulation in leaf 1 (CAL1), which encodes a defensin-like protein. CAL1 is expressed preferentially in root exodermis and xylem parenchyma cells. We provide evidence that CAL1 acts by chelating Cd in the cytosol and facilitating Cd secretion to extracellular spaces, hence lowering cytosolic Cd concentration while driving long-distance Cd transport via xylem vessels. CAL1 does not appear to affect Cd accumulation in rice grains or the accumulation of other essential metals, thus providing an efficient molecular tool to breed dual-function rice varieties that produce safe grains while remediating paddy soils.

Control of proline accumulation under drought via a novel pathway comprising the histone methylase CAU1 and the transcription factor ANAC055.

Proline plays a crucial role in the drought stress response in plants. However, there are still gaps in our knowledge about the molecular mechanisms that regulate proline metabolism under drought stress. Here, we report that the histone methylase encoded by CAU1, which is genetically upstream of P5CS1 (encoding the proline biosynthetic enzyme Δ1-pyrroline-5-carboxylate synthetase 1), plays a crucial role in proline-mediated drought tolerance. We determined that the transcript level of CAU1 decreased while that of ANAC055 (encoding a transcription factor) increased in wild-type Arabidopsis under drought stress. Further analyses showed that CAU1 bound to the promoter of ANAC055 and suppressed its expression via H4R3sme2-type histone methylation in the promoter region. Thus, under drought stress, a decreased level of CAU1 led to an increased transcript level of ANAC055, which induced the expression of P5CS1 and increased proline level independently of CAS. Drought tolerance and the level of proline were found to be decreased in the cau1 anac055 double-mutant, while proline supplementation restored drought sensitivity in the anac055 mutant. Our results reveal the details of a novel pathway leading to drought tolerance mediated by CAU1.

Arabidopsis NRT1.5 Mediates the Suppression of Nitrate Starvation-Induced Leaf Senescence by Modulating Foliar Potassium Level.

Nitrogen deficiency induces leaf senescence. However, whether or how nitrate might affect this process remains to be investigated. Here, we report an interesting finding that nitrate-instead of nitrogen-starvation induced early leaf senescence in nrt1.5 mutant, and present genetic and physiological data demonstrating that nitrate starvation-induced leaf senescence is suppressed by NRT1.5. NRT1.5 suppresses the senescence process dependent on its function from roots, but not the nitrate transport function. Further analyses using nrt1.5 single and nia1 nia2 nrt1.5-4 triple mutant showed a negative correlation between nitrate concentration and senescence rate in leaves. Moreover, when exposed to nitrate starvation, foliar potassium level decreased in nrt1.5, but adding potassium could essentially restore the early leaf senescence phenotype of nrt1.5 plants. Nitrate starvation also downregulated the expression of HAK5, RAP2.11, and ANN1 in nrt1.5 roots, and appeared to alter potassium level in xylem sap from nrt1.5. These data suggest that NRT1.5 likely perceives nitrate starvation-derived signals to prevent leaf senescence by facilitating foliar potassium accumulation.

LeNRT2.3 functions in nitrate acquisition and long-distance transport in tomato.

Nitrogen plays an important role in plant growth and development. Nitrate transporters have been extensively studied in Arabidopsis, but in tomato they have not been functionally characterized. In this study, we report the functions of LeNRT2.3 in nitrate transport in tomato. Our results show that LeNRT2.3 is induced by nitrate, and mainly localizes to the plasma membranes of rhizodermal and pericycle cells in roots. Further analysis in Xenopus oocytes showed that LeNRT2.3 mediates low-affinity nitrate transport. 35S:LeNRT2.3 increased nitrate uptake in root and transport from root to shoot. More interestingly, 35S:LeNRT2.3 showed high biomass and fruit weight. Taken together, these results suggest that LeNRT2.3 plays a double role in nitrate uptake and long-distance transport in tomato.

Arabidopsis histone methylase CAU1/PRMT5/SKB1 acts as an epigenetic suppressor of the calcium signaling gene CAS to mediate stomatal closure in response to extracellular calcium.

Elevations in extracellular calcium ([Ca(2+)]o) are known to stimulate cytosolic calcium ([Ca(2+)]cyt) oscillations to close stomata. However, the underlying mechanisms regulating this process remain largely to be determined. Here, through the functional characterization of the calcium underaccumulation mutant cau1, we report that the epigenetic regulation of CAS, a putative Ca(2+) binding protein proposed to be an external Ca(2+) sensor, is involved in this process. cau1 mutant plants display increased drought tolerance and stomatal closure. A mutation in CAU1 significantly increased the expression level of the calcium signaling gene CAS, and functional disruption of CAS abolished the enhanced drought tolerance and stomatal [Ca(2+)]o signaling in cau1. Map-based cloning revealed that CAU1 encodes the H4R3sme2 (for histone H4 Arg 3 with symmetric dimethylation)-type histone methylase protein arginine methytransferase5/Shk1 binding protein1. Chromatin immunoprecipitation assays showed that CAU1 binds to the CAS promoter and modulates the H4R3sme2-type histone methylation of the CAS chromatin. When exposed to elevated [Ca(2+)]o, the protein levels of CAU1 decreased and less CAU1 bound to the CAS promoter. In addition, the methylation level of H4R3sme2 decreased in the CAS chromatin. Together, these data suggest that in response to increases in [Ca(2+)]o, fewer CAU1 protein molecules bind to the CAS promoter, leading to decreased H4R3sme2 methylation and consequent derepression of the expression of CAS to mediate stomatal closure and drought tolerance.

The Arabidopsis nitrate transporter NRT1.8 functions in nitrate removal from the xylem sap and mediates cadmium tolerance.

Long-distance transport of nitrate requires xylem loading and unloading, a successive process that determines nitrate distribution and subsequent assimilation efficiency. Here, we report the functional characterization of NRT1.8, a member of the nitrate transporter (NRT1) family in Arabidopsis thaliana. NRT1.8 is upregulated by nitrate. Histochemical analysis using promoter-beta-glucuronidase fusions, as well as in situ hybridization, showed that NRT1.8 is expressed predominantly in xylem parenchyma cells within the vasculature. Transient expression of the NRT1.8:enhanced green fluorescent protein fusion in onion epidermal cells and Arabidopsis protoplasts indicated that NRT1.8 is plasma membrane localized. Electrophysiological and nitrate uptake analyses using Xenopus laevis oocytes showed that NRT1.8 mediates low-affinity nitrate uptake. Functional disruption of NRT1.8 significantly increased the nitrate concentration in xylem sap. These data together suggest that NRT1.8 functions to remove nitrate from xylem vessels. Interestingly, NRT1.8 was the only nitrate assimilatory pathway gene that was strongly upregulated by cadmium (Cd(2+)) stress in roots, and the nrt1.8-1 mutant showed a nitrate-dependent Cd(2+)-sensitive phenotype. Further analyses showed that Cd(2+) stress increases the proportion of nitrate allocated to wild-type roots compared with the nrt1.8-1 mutant. These data suggest that NRT1.8-regulated nitrate distribution plays an important role in Cd(2+) tolerance.

Genetic interactions between leaf polarity-controlling genes and ASYMMETRIC LEAVES1 and 2 in Arabidopsis leaf patterning.

During leaf development, establishment of adaxial-abaxial polarity is essential for normal leaf morphogenesis. This process is known to be strictly regulated by several putative transcription factors, microRNA165/166 (miR165/166), a trans-acting short-interfering RNA (tasiR-ARF), as well as proteins involved in RNA silencing. Among the putative transcription factor genes, ASYMMETRIC LEAVES1 and 2 (AS1 and 2) facilitate the specification of leaf adaxial identity; however, the mechanism by which AS1 and AS2 cooperate with other leaf polarity components remains largely undetermined. In the current study, we characterized the phenotype of mutants by combining as1 and as2 with mutations of several key transcription factors. Our data showed that double mutant plants carrying as1/as2 and rev, phb or phv enhanced as1/as2 defects by producing more severely abaxialized leaves. In contrast, triple mutants, obtained by combining as1/as2 with double mutant filamentous flower yabby3 (fil yab3) or kanadi1 kanadi2 (kan1 kan2), exhibited additive phenotypes. Additionally, while leaves of rev as2 contained high levels of FIL transcripts, only slightly elevated miR165/166 levels were noted, indicating that FIL and miR165/166 act in parallel in leaf patterning. Moreover, 35S::MIR165a/rev as2 transgenic plants resulted in a more severe abaxialized leaf phenotype than the rev and as2 single mutant plants transformed with the same 35S::MIR165a fusion. Genetic interactions between the key regulators during leaf patterning are discussed.

ASYMMETRIC LEAVES1, an Arabidopsis gene that is involved in the control of cell differentiation in leaves.

During leaf development, the formation of dorsal-ventral and proximal-distal axes is central to leaf morphogenesis. To investigate the genetic basis of dorsoventrality and proximodistality in the leaf, we screened for mutants of Arabidopsis thaliana (L.) Heynh. with defects in leaf morphogenesis. We describe here the phenotypic analysis of three mutant alleles that we have isolated. These mutants show varying degrees of abnormality including dwarfism, broad leaf lamina, and aberrant floral organs and fruits. Genetic analysis revealed that these mutations are alleles of the previously isolated mutant asymmetric leaves1 ( as1). In addition to the leaf phenotypes described previously, these alleles display other phenotypes that were not observed. These include: (i) some rosette leaves with petiole growth underneath the leaf lamina; (ii) leaf vein branching in the petiole; and (iii) a leaf lamina with an epidermis similar to that on the petiole. The mutant phenotypes suggest that the ASYMMETRIC LEAVES1 ( AS1) gene is involved in the control of cell differentiation in leaves. As the first step in determining a molecular function for AS1, we have identified the AS1 gene using map-based cloning. The AS1 gene encodes a MYB-domain protein that is homologous to the Antirrhinum PHANTASTICA ( PHAN) and maize ROUGH SHEATH2 ( RS2) genes. AS1 is expressed nearly ubiquitously, consistent with the pleiotropic mutant phenotypes. High levels of AS1 expression were found in tissues with highly proliferative cells, which further suggests a role in cell division and early cell differentiation.