Gel-based proteomics approach for detecting low nitrogen-responsive proteins in cultivated rice species

Abstract

Nitrogen fertilization is essential for increasing rice production to meet the food demands of increasing world's population. We established an in vivo hydroponic rice seedling culture system to investigate physio-biochemical/molecular responses of various rice japonica and indica cultivars to low nitrogen (N). Three-week-old seedlings grown in Yoshida's nutrient solution manifested stable and reproducible symptoms, such as reduced shoot growth and length under low N. Out of 12 genetically selected cultivars, 11 cultivars showed varied degrees of growth reduction response to applied N (4 and 40 ppm N for treatment and control, respectively), whereas one cultivar (no. 12) showed similar growth as the control though its leaf width was smaller than control. Leaves of a representative low N-responsive cultivar (BG90-2) were sampled for revealing protein profiles between low and normal (control) N application by two-dimensional gel electrophoresis (2-DGE). Forty-one proteins were identified with MALDI-TOF-MS and nESI-LC-MS/MS. Assignment of proteins into major (energy metabolism, photosynthesis and oxidative stress) and minor functional categories, revealed many novel low N-responsive proteins, including those having energy/photosynthesis-and defense/stress-and iron homeostasis-related functions. Results suggest the usefulness of proteomics in identifying novel N-responsive proteins and may provide potential markers for rice response to low N.

Proteomics dissection of plant responses to mineral nutrient deficiency

“…To cope with N deficiency, plants have developed multiple levels of strategies to increase N acquisition and use efficiency, which have been well illustrated through physiological, genetic, and molecular studies [5,6,35,36]. Proteomic analysis of proteins responsive to short-and long-term N deficiency was also performed in plants, including maize, rice, Arabidopsis, triticale (Triticosecale wittmack), wheat (Triticum aestivum), barley (Hordeum vugare), tobacco (Nicotiana tabacum), and creeping bentgrass (Agrostis tolonifera) through 2-D, followed by MS analysis [37][38][39][40][41][42][43][44][45][46][47][48][49], which was briefly summarized (Fig. 2, Table 2).…”

“…symptoms of N deficiency were obviously observed) in leaves and roots ( Table 2). Results showed that most responsive proteins identified from leaves and roots were distinct, suggesting leaves and roots might exhibit different adaptive strategies to longterm N deficiency [37][38][39][40][41][42][43][44][45][46][47][48][49].…”

“…Long-term N deficiency generally results in pale green leaf color in plants. Thus, most of the N deficiency responsive proteins in leaves were involved in photosynthesis, and were obviously down-regulated, such as ATP synthase beta subunit, RuBisCO activase, subunits of photosystem, ferredoxin-NADP reductase, and RuBisCO large and small subunit [38,41,45,46]. The suppressed accumulations of these proteins correlated well with the lower rates of photosynthesis under long-term N deficiency, suggesting that inhibited plant growth and yield by N deficiency was mainly caused by the inhibition of photosynthesis [50,51].…”

“…The suppressed accumulations of these proteins correlated well with the lower rates of photosynthesis under long-term N deficiency, suggesting that inhibited plant growth and yield by N deficiency was mainly caused by the inhibition of photosynthesis [50,51]. On the other hand, several proteins involved in photosynthesis were found to be up-regulated by N deficiency, such as carbonic anhydrases and type II light-harvesting chlorophyll a/b-binding protein in rice [42], O-evolving enhancer protein [39,41] and a 23 kD polypeptide photosystem II in rice and maize [39,41]. Increased accumulations of these proteins might be a compensated response for the inhibited photosynthesis to avoid severely adverse effects of N deficiency on plant growth.…”

“…Compared to photosynthesis and photorespiration, N deficiency results in more complex responses of C metabolism in leaves, as reflected by the changes of many C metabolism related proteins in plants under N-deficient conditions [38,41,42,45,49]. For example, in leaves of rice, several proteins were up-regulated under N-deficient conditions, such as fructose 1,6-bisphosphate aldolase, dihydrolipoamide dehydrogenase precursor, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerases, and carbonate anhydrase [41,42]. Similar proteins were also found to be accumulated in triticale and barley subjected to N deficiency [45,49], suggesting the coordinated alterations of these proteins might be essential for the activation of the entire energy-producing pathway to support the N-deficient plants.…”