Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC-CONICET), Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, X5000HUA, Córdoba, Argentina.
Ann Bot. 2013 Nov;112(7):1351-60. doi: 10.1093/aob/mct207. Epub 2013 Sep 18.
Non-S-ribonucleases (non-S-RNases) are class III T2 RNases constitutively expressed in styles of species with S-RNase-based self-incompatibility. So far, no function has been attributed to these RNases. The aim of this work is to examine if NnSR1, a non-S-RNase from Nicotiana alata, is induced under conditions of phosphate (Pi) deprivation. The hypothesis is that under Pi-limited conditions, non-S-RNase functions may resemble the role of S-like RNases. To date, the only RNases reported to be induced by Pi deficiency are class I and class II S-like RNases, which are phylogenetically different from the class III clade of RNases.
Gene and protein expression of NnSR1 were assayed in plants grown hydroponically with and without Pi, by combining RT-PCR, immunoblot and enzymatic activity approaches.
NnSR1 transcripts were detected in roots 7 d after Pi deprivation and remained stable for several days. Transcript expression was correlated based on Pi availability in the culture medium. Antiserum against a peptide based on a hypervariable domain of NnSR1 recognized NnSR1 in roots and stems but not leaves exposed to Pi shortage. NnSR1 was not detected in culture medium and was pelleted with the microsomal fraction, suggesting that it was membrane-associated or included in large compartments. The anti-NnSR1 inhibited selectively the enzymatic activity of a 31-kDa RNase indicating that NnSR1 was induced in an enzymatically active form.
The induction of NnSR1 indicates that there is a general recruitment of all classes of T2 RNases in response to Pi shortage. NnSR1 appears to have regained ancestral functions of class III RNases related to strategies to cope with Pi limitation and also possibly with other environmental challenges. This constitutes the first report for a specific function of class III RNases other than S-RNases.
非 S-核糖核酸酶（non-S-RNases）是在具有 S-RNase 为基础的自交不亲和性的物种的花柱中组成型表达的 III 类 T2 RNases。到目前为止，还没有赋予这些 RNases 任何功能。这项工作的目的是研究来自 Nicotiana alata 的非 S-RNase NnSR1 是否在磷酸盐（Pi）剥夺的条件下被诱导。假设是在 Pi 有限的条件下，非 S-RNase 的功能可能类似于 S 类 RNases 的作用。迄今为止，据报道，只有 I 类和 II 类 S 类 RNases是由 Pi 缺乏诱导的，它们在系统发育上与 RNase 的 III 类分支不同。
通过结合 RT-PCR、免疫印迹和酶活性方法，在有和没有 Pi 的水培条件下，检测 NnSR1 基因和蛋白质的表达。
在 Pi 缺乏 7 天后，在根中检测到 NnSR1 转录物，并在几天内保持稳定。基于培养基中 Pi 的可用性，转录物表达呈相关性。针对 NnSR1 高变区的肽的抗血清在暴露于 Pi 缺乏的根和茎中识别 NnSR1，但不在叶中识别。NnSR1 未在培养基中检测到，并与微粒体部分一起沉淀，表明它是膜相关的或包含在大的隔室中。抗 NnSR1 选择性抑制 31 kDa RNase 的酶活性，表明 NnSR1 以酶活性形式被诱导。
NnSR1 的诱导表明，所有 T2 RNase 类都普遍参与了对 Pi 缺乏的反应。NnSR1 似乎重新获得了与应对 Pi 限制和可能其他环境挑战相关的 III 类 RNase 的祖先功能。这是除 S-RNases 之外，III 类 RNases 具有特定功能的首次报道。
