Physiological and transcriptomic analyses reveal tea plant (Camellia sinensis L.) adapts to extreme freezing stress during winter by regulating cell wall structure.

Tea plants grown in high-latitude areas are often damaged by extreme freezing temperatures in winter, leading to huge economic losses. Here, the physiological and gene expression characteristics of two tea cultivars (Xinyang No. 10 (XY10), a freezing-tolerant cultivar and Fudingdabaicha (FDDB), a freezing-sensitive cultivar) during overwintering in northern China were studied to better understand the regulation mechanisms of tea plants in response to natural freezing stress. Samples were collected at a chill (D1), freezing (D2) and recovery (D3) temperature in winter. TEM analysis of integrated leaf ultrastructure at D2 revealed lower malondialdehyde and relative electrical conductivity in XY10 than in FDDB, with serious cell structure damage in the latter, indicating XY10 was more resistant to freezing stress. Differential gene expression analysis among the different samples over winter time highlighted the following gene functions in cell wall metabolism (CesAs, COBLs, XTHs, PGs, PMEs), transcription factors (ERF1B and MYC2), and signal transduction (CDPKs and CMLs). The expression pattern of cellulose and pectin-related genes suggested higher accumulation of cellulosic and pectic materials in the cell wall of XY10, agreeing with the results of cell wall and its components. These results indicated that under the regulation of cell wall genes, the freezing-resistant tea cultivar can better maintain a well-knit cell wall structure with sufficient substances to survive natural freezing damage. This study demonstrated the crucial role of cell wall in tea plant resistance to natural freezing stress and provided important candidate genes for breeding of freezing-resistant tea cultivars.

A Transcription Factor, OsMADS57, Regulates Long-Distance Nitrate Transport and Root Elongation.

Root nitrate uptake adjusts to the plant's nitrogen demand for growth. Here, we report that OsMADS57, a MADS-box transcription factor, modulates nitrate translocation from rice (Oryza sativa) roots to shoots under low-nitrate conditions. OsMADS57 is abundantly expressed in xylem parenchyma cells of root stele and is induced by nitrate. Compared with wild-type rice plants supplied with 0.2 mM nitrate, osmads57 mutants had 31% less xylem loading of nitrate, while overexpression lines had 2-fold higher levels. Shoot-root 15N content ratios were 40% lower in the mutants and 76% higher in the overexpression lines. Rapid NO3 - root influx experiments showed that mutation of OsMADS57 did not affect root nitrate uptake. Reverse transcription quantitative PCR analysis of OsNRT2 nitrate transporter genes showed that after 5 min in 0.2 mM nitrate, only OsNRT2.3a (a vascular-specific high-affinity nitrate transporter) had reduced (by two-thirds) expression levels. At 60 min of nitrate treatment, lower expression levels were also observed for three additional NRT2 genes (OsNRT2.1/2.2/2.4). Conversely, in the overexpression lines, four NRT2 genes had much higher expression profiles at all time points tested. As previously reported, OsNRT2.3a functions in nitrate translocation, indicating the possible interaction between OsMADS57 and OsNRT2.3a Yeast one-hybrid and transient expression assays demonstrated that OsMADS57 binds to the CArG motif (CATTTTATAG) within the OsNRT2.3a promoter. Moreover, seminal root elongation was inhibited in osmads57 mutants, which may be associated with higher auxin levels in and auxin polar transport to root tips of mutant plants. Taken together, these results suggest that OsMADS57 has a role in regulating nitrate translocation from root to shoot via OsNRT2.3a.

Strigolactones are required for nitric oxide to induce root elongation in response to nitrogen and phosphate deficiencies in rice.

The response of the root system architecture to nutrient deficiencies is critical for sustainable agriculture. Nitric oxide (NO) is considered a key regulator of root growth, although the mechanisms remain unknown. Phenotypic, cellular and genetic analyses were undertaken in rice to explore the role of NO in regulating root growth and strigolactone (SL) signalling under nitrogen-deficient and phosphate-deficient conditions (LN and LP). LN-induced and LP-induced seminal root elongation paralleled NO production in root tips. NO played an important role in a shared pathway of LN-induced and LP-induced root elongation via increased meristem activity. Interestingly, no responses of root elongation were observed in SL d mutants compared with wild-type plants, although similar NO accumulation was induced by sodium nitroprusside (SNP) application. Application of abamine (the SL inhibitor) reduced seminal root length and pCYCB1;1::GUS expression induced by SNP application in wild type; furthermore, comparison with wild type showed lower SL-signalling genes in nia2 mutants under control and LN treatments and similar under SNP application. Western blot analysis revealed that NO, similar to SL, triggered proteasome-mediated degradation of D53 protein levels. Therefore, we presented a novel signalling pathway in which NO-activated seminal root elongation under LN and LP conditions, with the involvement of SLs.

Knockdown of the partner protein OsNAR2.1 for high-affinity nitrate transport represses lateral root formation in a nitrate-dependent manner.

The morphological plasticity of root systems is critical for plant survival, and understanding the mechanisms underlying root adaptation to nitrogen (N) fluctuation is critical for sustainable agriculture; however, the molecular mechanism of N-dependent root growth in rice remains unclear. This study aimed to identify the role of the complementary high-affinity NO3(-) transport protein OsNAR2.1 in NO3(-)-regulated rice root growth. Comparisons with wild-type (WT) plants showed that knockdown of OsNAR2.1 inhibited lateral root (LR) formation under low NO3(-) concentrations, but not under low NH4(+) concentrations. (15)N-labelling NO3(-) supplies (provided at concentrations of 0-10 mM) demonstrated that (i) defects in LR formation in mutants subjected to low external NO3(-) concentrations resulted from impaired NO3(-) uptake, and (ii) the mutants had significantly fewer LRs than the WT plants when root N contents were similar between genotypes. LR formation in osnar2.1 mutants was less sensitive to localised NO3(-) supply than LR formation in WT plants, suggesting that OsNAR2.1 may be involved in a NO3(-)-signalling pathway that controls LR formation. Knockdown of OsNAR2.1 inhibited LR formation by decreasing auxin transport from shoots to roots. Thus, OsNAR2.1 probably functions in both NO3(-) uptake and NO3(-)-signalling.

A strigolactone signal is required for adventitious root formation in rice.

BACKGROUND AND AIMS: Strigolactones (SLs) and their derivatives are plant hormones that have recently been identified as regulating root development. This study examines whether SLs play a role in mediating production of adventious roots (ARs) in rice (Oryza sativa), and also investigates possible interactions between SLs and auxin. METHODS: Wild-type (WT), SL-deficient (d10) and SL-insensitive (d3) rice mutants were used to investigate AR development in an auxin-distribution experiment that considered DR5::GUS activity, [(3)H] indole-3-acetic acid (IAA) transport, and associated expression of auxin transporter genes. The effects of exogenous application of GR24 (a synthetic SL analogue), NAA (α-naphthylacetic acid, exogenous auxin) and NPA (N-1-naphthylphalamic acid, a polar auxin transport inhibitor) on rice AR development in seedlings were investigated. KEY RESULTS: The rice d mutants with impaired SL biosynthesis and signalling exhibited reduced AR production compared with the WT. Application of GR24 increased the number of ARs and average AR number per tiller in d10, but not in d3. These results indicate that rice AR production is positively regulated by SLs. Higher endogenous IAA concentration, stronger expression of DR5::GUS and higher [(3)H] IAA activity were found in the d mutants. Exogenous GR24 application decreased the expression of DR5::GUS, probably indicating that SLs modulate AR formation by inhibiting polar auxin transport. The WT and the d10 and d3 mutants had similar expression of DR5::GUS regardless of exogenous application of NAA or NPA; however, AR number was greater in the WT than in the d mutants. CONCLUSIONS: The results suggest that AR formation is positively regulated by SLs via the D3 response pathway. The positive effect of NAA application and the opposite effect of NPA application on AR number of WT plants also suggests the importance of auxin for AR formation, but the interaction between auxin and SLs is complex.

Nitric oxide generated by nitrate reductase increases nitrogen uptake capacity by inducing lateral root formation and inorganic nitrogen uptake under partial nitrate nutrition in rice.

Increasing evidence shows that partial nitrate nutrition (PNN) can be attributed to improved plant growth and nitrogen-use efficiency (NUE) in rice. Nitric oxide (NO) is a signalling molecule involved in many physiological processes during plant development and nitrogen (N) assimilation. It remains unclear whether molecular NO improves NUE through PNN. Two rice cultivars (cvs Nanguang and Elio), with high and low NUE, respectively, were used in the analysis of NO production, nitrate reductase (NR) activity, lateral root (LR) density, and (15)N uptake under PNN, with or without NO production donor and inhibitors. PNN increased NO accumulation in cv. Nanguang possibly through the NIA2-dependent NR pathway. PNN-mediated NO increases contributed to LR initiation, (15)NH4(+)/(15)NO3(-) influx into the root, and levels of ammonium and nitrate transporters in cv. Nanguang but not cv. Elio. Further results revealed marked and specific induction of LR initiation and (15)NH4(+)/(15)NO3(-) influx into the roots of plants supplied with NH4(+)+sodium nitroprusside (SNP) relative to those supplied with NH4(+) alone, and considerable inhibition upon the application of cPTIO or tungstate (NR inhibitor) in addition to PNN, which is in agreement with the change in NO fluorescence in the two rice cultivars. The findings suggest that NO generated by the NR pathway plays a pivotal role in improving the N acquisition capacity by increasing LR initiation and the inorganic N uptake rate, which may represent a strategy for rice plants to adapt to a fluctuating nitrate supply and increase NUE.

Strigolactones are involved in phosphate- and nitrate-deficiency-induced root development and auxin transport in rice.

Strigolactones (SLs) or their derivatives have recently been defined as novel phytohormones that regulate root development. However, it remains unclear whether SLs mediate root growth in response to phosphorus (P) and nitrogen (N) deficiency. In this study, the responses of root development in rice (Oryza sativa L.) to different levels of phosphate and nitrate supply were investigated using wild type (WT) and mutants defective in SL synthesis (d10 and d27) or insensitive to SL (d3). Reduced concentration of either phosphate or nitrate led to increased seminal root length and decreased lateral root density in WT. Limitation of either P or N stimulated SL production and enhanced expression of D10, D17, and D27 and suppressed expression of D3 and D14 in WT roots. Mutation of D10, D27, or D3 caused loss of sensitivity of root response to P and N deficiency. Application of the SL analogue GR24 restored seminal root length and lateral root density in WT and d10 and d27 mutants but not in the d3 mutant, suggesting that SLs were induced by nutrient-limiting conditions and led to changes in rice root growth via D3. Moreover, P or N deficiency or GR24 application reduced the transport of radiolabelled indole-3-acetic acid and the activity of DR5::GUS auxin reporter in WT and d10 and d27 mutants. These findings highlight the role of SLs in regulating rice root development under phosphate and nitrate limitation. The mechanisms underlying this regulatory role involve D3 and modulation of auxin transport from shoots to roots.

Auxin distribution is differentially affected by nitrate in roots of two rice cultivars differing in responsiveness to nitrogen.

BACKGROUND AND AIMS: Although ammonium (NH4(+)) is the preferred form of nitrogen over nitrate (NO3(-)) for rice (Oryza sativa), lateral root (LR) growth in roots is enhanced by partial NO3(-) nutrition (PNN). The roles of auxin distribution and polar transport in LR formation in response to localized NO3(-) availability are not known. METHODS: Time-course studies in a split-root experimental system were used to investigate LR development patterns, auxin distribution, polar auxin transport and expression of auxin transporter genes in LR zones in response to localized PNN in 'Nanguang' and 'Elio' rice cultivars, which show high and low responsiveness to NO3(-), respectively. Patterns of auxin distribution and the effects of polar auxin transport inhibitors were also examined in DR5::GUS transgenic plants. KEY RESULTS: Initiation of LRs was enhanced by PNN after 7 d cultivation in 'Nanguang' but not in 'Elio'. Auxin concentration in the roots of 'Nanguang' increased by approx. 24 % after 5 d cultivation with PNN compared with NH4(+) as the sole nitrogen source, but no difference was observed in 'Elio'. More auxin flux into the LR zone in 'Nanguang' roots was observed in response to NO3(-) compared with NH4(+) treatment. A greater number of auxin influx and efflux transporter genes showed increased expression in the LR zone in response to PNN in 'Nanguang' than in 'Elio'. CONCLUSIONS: The results indicate that higher NO3(-) responsiveness is associated with greater auxin accumulation in the LR zone and is strongly related to a higher rate of LR initiation in the cultivar 'Nanguang'.