The Effect of the Crosstalk between Photoperiod and Temperature on the Heading-Date in Rice

Photoperiod and temperature are two important environmental factors that influence the heading-date of rice. Although the influence of the photoperiod on heading has been extensively reported in rice, the molecular mechanism for the temperature control of heading remains unknown. This study reports an early heading mutant derived from tissue culture lines of rice and investigates the heading-date of wild type and mutant in different photoperiod and temperature treatments. The linkage analysis showed that the mutant phenotype cosegregated with the Hd1 locus. Sequencing analysis found that the mutant contained two insertions and several single-base substitutions that caused a dramatic reduction in Hd1mRNA levels compared with wild type. The expression patterns of Hd1 and Hd3a were also analyzed in different photoperiod and temperature conditions, revealing that Hd1 mRNA levels displayed similar expression patterns for different photoperiod and temperature treatments, with high expression levels at night and reduced levels in the daytime. In addition, Hd1 displayed a slightly higher expression level under long-day and low temperature conditions. Hd3a mRNA was present at a very low level under low temperature conditions regardless of the day-length. This result suggests that suppression of Hd3a expression is a principle cause of late heading under low temperature and long-day conditions

Sequence analysis of <i>Hd1</i> in <i>lf1132</i> and wild type.

<p>A: The sequence differences between wild type and <i>lf1132</i>. The black triangle represents insertion; vertical lines represent single-base substitutions; blue vertical lines and numbers are relative positions in <i>hd1-3</i>. SEF and SER shown by arrows are primers to detect the 315 bp insertion. B: PCR detection of the 315 bp insertion on the <i>hd1-3</i> locus for <i>lf1132</i>. C: The expression of <i>Hd1</i> in the wild type and mutant. Leaves were harvested from 30 day old seedlings at the indicated times (once every 3 h for 24 h) in natural fields (day-length is about 14 h light and 10 h dark) and RT-PCR was carried out for the analysis of <i>Hd1</i> expression. Primer pairs HD1F and HD1R were used for the analysis of <i>Hd1</i> expression in RT-PCR. D: Deduced amino acid sequence of the Hd1 and deduced lf1132 proteins. The black line indicates the zinc-finger domain; asterisks are amino acid substitutions between the Nipponbare Hd1 protein and the deduced lf1132 protein. E: the linkage analysis of the mutant and <i>Hd1</i> locus. P₁ is Zhonghua 11; P₂ is <i>lf1132</i>.</p

The phenotype of the mutant.

<p>A: the phenotype of the mutant and wild type, M is mutant <i>lf1132</i>; WT is Zhonghua 11. B: the panicle length and internode length for mutants and wild type. 15 total plants were investigated from five repeats containing three individuals. C: The heading-date of the wild type and mutant on different sowing-dates. Wild type and mutant were planted in the CNRRI experimental field, Zhejiang province on six sowing-dates from 15, May to 21, July 2007. D: The change in photoperiod during different sowing-dates. E: The change in temperature (mean value of everyday temperature) during different sowing-dates. Red box indicates the temperature of the heading period at the last sowing-date, 21, July.</p

The heading-date of the mutant and wild type for different photoperiod and temperature treatments.

<p><i>lf1132</i> and wild type plants were planted in the CNRRI experimental fields, and two week old seedlings were transferred to phytotrons with different photoperiod and temperature treatments. The heading-date for each treatment was observed and recorded for at least 10 plants. Four phytotrons were used: LD, 27°C phytotron; LD, 23°C phytotron; SD, 27°C phytotron; SD, 23°C phytotron; A: the heading-date under different photoperiods and temperatures; B: The velocity ratio of leaf growth (VRL) for the mutant and wild type under different photoperiods (SD and LD) and temperatures (27°C, 23°C). LD treatment: 14.5 h light and 9.5 h dark; SD treatment: 11.5 h light and 12.5 h dark.</p

<i>Hd3a</i> expression under different photoperiods and temperatures.

<p>Leaves were harvested from 33 day old plants at the indicated times (once every 4 h for 24 h) in phytotrons, and real-time PCR was carried out for the analysis of <i>Hd3a</i> expression. M is <i>lf1132</i>; WT is Zhonghua 11. A, B, C, D are the <i>Hd3a</i> expression profiles under high temperature and low temperature; A: wild type under LD condition; B: wild type under SD condition; C: mutant under LD condition; D: mutant under SD condition. E and F are the <i>Hd3a</i> expression profiles for the wild type and mutant under different photoperiods at high temperature; E: wild type; F: mutant. G presents the <i>Hd3a</i> expression profile of the mutant and wild type under LD conditions.</p

<i>Hd1</i> expression under different photoperiods and temperatures.

<p>Leaves were harvested from 33 day old plants at the indicated times (once every 4 h for 24 h) in phytotrons, and real-time PCR was carried out for analysis of <i>Hd1</i>. M is <i>lf1132</i>; WT is Zhonghua 11.</p

Additional file 1 of OsNAC103, a NAC Transcription Factor, Positively Regulates Leaf Senescence and Plant Architecture in Rice

Additional file 1. Fig. S1: Phylogenic analysis and sequence analysis of OsNAC103. A An unrooted phylogenetic tree of stress-responsive NAC (SNAC) proteins in rice and Arabidopsis. SNAC-A and -B are two subgroups of SNAC proteins. The tree was drawn using the Neighbor-Joining method in the MEGA 11.0 program. B Multiple sequence alignments between OsNAC103 and other members of the NAC subfamily in rice. (a)–(e) represent five highly conservative regions. Fig. S2: Phenotype and overexpression levels of OsNAC103-OE lines in T0 generation. A Phenotypes of OsNAC103-OE lines. B Overexpression levels of OsNAC103-OE lines using qRT-PCR. OE1, OE2, OE6, OE7 and OE9 are five independent OsNAC103-OE lines. Asterisks indicate statistically significant differences by Student’s t test (*, P < 0.05; **, P < 0.01). Fig. S3: Sequencing analysis of osnac103 mutants (CR2 and CR5) by CRISPR-Cas9 system. A Target sites of CRISPR-Cas9 for OsNAC103. Solid boxes, exons; hollow box, 5′-UTR; hollow pentagon, 3′-UTR; the lines, introns; Target1, Target2 and Target3 represent three targets of OsNAC103, respectively. B–G Mutation sites of CR2 and CR5. Red boxes mean the position of mutations.—means deletion. Red underlines mean the position of PAM. Fig. S4: OsNAC103 positively regulates leaf senescence in rice. A–C Phenotype of OsNAC103-OE lines and osnac103 mutants during the vegetative growth stage. Bars = 20 cm. B indicates the magnified figure in A. (C) Phenotype of OsNAC103-OE lines and osnac103 mutants in the field. D, E Phenotype and total chlorophyll contents of different leaves in OsNAC103-OE lines and osnac103 mutants. Leaf-2, Leaf-3, Leaf-4, Leaf-5 represent the second, third, fourth and fifth leaves of the rice plant from top down, respectively. Bar = 5 cm. OE2 and OE7 are two independent OsNAC103-OE lines. CR2 and CR5 are two allelic mutants. Values are shown as means ± SD, n = 3. Asterisks indicate statistically significant differences by Student’s t test (*, P < 0.05; **, P < 0.01). Fig. S5: Agronomic traits of OsNAC103-OE lines and osnac103 mutants. A Panicle length, number of primary branches and 100 grains weight of OsNAC103-OE lines and osnac103 mutants. Values are shown as means ± SD, n ≥ 10 individual plants. Asterisks indicate statistically significant differences by Student’s t test (*, P < 0.05; **, P < 0.01). Fig. S6: Expression analysis of genes associated with tiller angle and shoot gravitropism in OsNAC103-OE lines and osnac103 mutants. RNAs were extracted from penultimate leaves of WT, OE lines and osnac103 mutants at vegetative stage, and qRT-PCR was performed to analyze the relative expression levels of different genes. OE2 and OE7 are two independent OsNAC103-OE lines. CR2 and CR5 are two allelic mutants. Asterisks indicate statistically significant differences by Student’s t test (*, P < 0.05; **, P < 0.01). Fig. S7: Interaction of OsNAC103 and the promoters of leaf senescence-associated genes. Yeast cells transformed with the indicated plasmids were grown on selective SD/-Ura/-Leu medium added with X-Gal. The interaction of pLacZi-proOsMADS14 and OsFTL12 effector was as a positive control. Fig. S8: Alternative splices of OsNAC103 and the blast of their amino acid sequences. A Three alternative splicing forms of OsNAC103 indicated as Loc_Os07g48450.1, Loc_Os07g48450.2 and Loc_Os07g48450.3, respectively. Solid boxes, exons; hollow box, 5′-UTR; hollow pentagon, 3′-UTR; the lines, introns. B Alignment of amino acids sequence for the Loc_Os07g48450.1, Loc_Os07g48450.2 and Loc_Os07g48450.3. C RT-PCR analysis of different transcripts of Loc_Os07g48450. cDNA was obtained from leaves at ripening stage by the reverse transcription reaction. RT-PCR were performed to analyze the possible transcripts (indicated by red arrows). F, R and R’ were different primers indicated in (A). M1 and M2 were DNA ladder

The effect of different photoperiods and temperatures on heading-date.

<p>Notes: Zhonghua 11 and <i>lf1132</i> were grown in phytotrons with four different treatments. Heading-date was investigated at least 10 plants for each treatment.</p