Plants need abundant nitrogen and phosphorus for higher yield. Improving plant genetics for higher nitrogen and phosphorus use efficiency would save potentially billions of dollars annually on fertilizers and reduce global environmental pollution. This will require knowledge of molecular regulators for maintaining homeostasis of these nutrients in plants. Previously, we reported that the NITROGEN LIMITATION ADAPTATION (NLA) gene is involved in adaptive responses to low-nitrogen conditions in Arabidopsis, where nla mutant plants display abrupt early senescence. To understand the molecular mechanisms underlying NLA function, two suppressors of the nla mutation were isolated that recover the nla mutant phenotype to wild type. Map-based cloning identified these suppressors as the phosphate (Pi) transport-related genes PHF1 and PHT1.1. In addition, NLA expression is shown to be regulated by the low-Pi induced microRNA miR827. Pi analysis revealed that the early senescence in nla mutant plants was due to Pi toxicity. These plants accumulated over five times the normal Pi content in shoots specifically under low nitrate and high Pi but not under high nitrate conditions. Also the Pi overaccumulator pho2 mutant shows Pi toxicity in a nitrate-dependent manner similar to the nla mutant. Further, the nitrate and Pi levels are shown to have an antagonistic crosstalk as displayed by their differential effects on flowering time. The results demonstrate that NLA and miR827 have pivotal roles in regulating Pi homeostasis in plants in a nitrate-dependent fashion.

SummaryAbundant nitrogen is required for the optimal growth and development of plants, while numerous biotic and abiotic factors that consume soil nitrogen frequently create a nitrogen limitation growth condition. To cope with this, plants have evolved a suite of adaptive responses to nitrogen limitation. However, the molecular mechanism governing the adaptability of plants to nitrogen limitation is totally unknown because no reported mutant defines this trait. Here we isolated an Arabidopsis mutant, nla (nitrogen limitation adaptation), and identified the NLA gene as an essential component in this molecular mechanism. Supplied with insufficient inorganic nitrogen (nitrate or ammonium), the nla mutant failed to develop the essential adaptive responses to nitrogen limitation, but senesced much earlier and more rapidly than did the wild type. Under other stress conditions including low phosphorus nutrient, drought and high temperature, the nla mutant did not show this early senescence phenotype, but closely resembled the wild type in growth and development. Map-based cloning of NLA revealed that this gene encodes a RING-type ubiquitin ligase, and nla is a deletion mutation which does not code for the RING domain in the NLA protein. The NLA protein is localized to the nuclear speckles, where this protein interacts with the Arabidopsis ubiquitin conjugase 8 (AtUBC8). In the nla mutant, the deletion of the RING domain from NLA altered its subcellular localization, disrupted the interaction between NLA and AtUBC8 and caused the early senescence phenotype induced by low inorganic nitrogen. All the results indicate that NLA is a positive regulator for the development of the adaptability of Arabidopsis to nitrogen limitation.

Mature wheat (Triticum aestivum L.) endosperm contains two types of starch granules: large A‐type and small B‐type. Two methods, microsieving or centrifugal sedimentation through aqueous solutions of sucrose, maltose, or Percoll were used to separate A‐ and B‐type starch granules. Microsieving could not completely separate the two types of starch granules, while centrifuging through maltose and sucrose solutions gave a homogenous population for B‐type starch granules only. Centrifuging through two Percoll solutions (70 and 100%, v/v) produced purified populations of both the A‐ and B‐type starch granules. Analysis of starch granule size distribution in the purified A‐ and B‐type granule populations and in the whole‐starch granule population obtained directly from wheat endosperm confirmed that the purified A‐ and B‐type starch granule populations represented their counterparts in mature wheat endosperm. Centrifugations through two Percoll solutions were used to purify A‐ and B‐type starch granule populations from six wheat cultivars. The amylose concentrations and gelatinization properties of these populations were analyzed. All of the A‐type starch granules contained higher amylose concentrations and had higher gelatinization enthalpies than did B‐type starch granules. Although A‐ and B‐type starch granules started to gelatinize at a similar temperature, B‐type starch granules had higher gelatinization peak and completion temperatures than did A‐type starch granules