Class II members of the poly(A) binding protein family exhibit distinct functions during Arabidopsis growth and development

Figures & data

Figure 1. Mutants with altered expression of the widely-expressed class II PABP isoforms. (A) Gene structure and location of the T-DNA insertion in PAB2, PAB4, and PAB8 genes. Bars indicate exons and bent lines indicate introns. The location of the T-DNA insertion in each class II PABP member is indicated by the arrow head. (B) Western analysis of class II single and double PABP mutants to detect PABP (top panel) and eIF4A (bottom panel). PAB4 and PAB8 comigrate whereas PAB2 migrates just below.

Figure 2. Growth characteristics of pab mutants. (A) Growth of class II single and double PABP mutants at 3.5 weeks just before the appearance of the inflorescence relative to wild-type plants. (B) All leaves from adult wild-type and mutant plants at the appearance of the inflorescence. (C) Mature siliques from adult wild-type and mutant plants.

Figure 3. The effect of pab mutants on inflorescence growth. Flowering wild-type and class II PABP mutant plants with the inflorescence displayed.

Figure 4. Flower, silique, and embryo development of pab mutants. Flowers (top panels) and dissected siliques (bottom panels) showing developing embryos are shown for wild-type and class II PABP mutants. Bar represents 5 mm.

Table 1. Requirement for class II PABP members during seed development.

	Seed/silique	% seed set	Siliques/plant	% silique set	Silique length
WT	45.9 ± 3.14	98.9	51.8 ± 6.85	98.7	14.9 ± 1.19
pab2	42.2 ± 7.90	99.1	49.3 ± 5.86	100	15.0 ± 0.77
pab4	47.5 ± 0.97	98.1	53.8 ± 4.27	99.5	16.5 ± 1.81
pab8	52.7 ± 2.91^**	98.6	54.8 ± 3.86	100	15.2 ± 0.58
pab2/8	25.5 ± 4.03^**	61.6^**	53.8 ± 8.06	98.3	13.2 ± 1.77^*
pab4/8	44.4 ± 5.62	99.0	56.0 ± 2.0	99.4	14.1 ± 1.17
pab2/4	21.6 ± 5.06^**	58.3^**	20.2 ± 2.77^**	98.9	9.53 ± 0.74^**

Notes.

* , P < 0.05;

** , P < 0.001.

Figure 5. Flower, silique, and embryo development of heterozygous pab mutants. Flowers (top panels) and dissected siliques (bottom panels) showing developing embryos are shown for wild-type and heterozygous class II PABP mutants. Bar represents 5 mm.

Table 2. Effect of heterozygous class II PABP members during seed development.

	Seed/silique	% seed set	Siliques/plant	% silique set	Silique length
WT	47.2 ± 1.93	98.9	51.8 ± 6.85	98.7	14.9 ± 1.19
PAB4+/−pab8	50.7 ± 3.96	98.6	48.3 ± 2.08	100	16.2 ± 0.56^**
PAB8+/−pab4	49.1 ± 3.23	99.5	55.3 ± 7.51	100	14.9 ± 1.38
PAB2+/−pab4	49.8 ± 3.51	98.3	50.0 ± 2.00	100	15.9 ± 0.62^*
PAB4+/−pab2	29.8 ± 3.11^**	70.8^**	38.8 ± 4.99^**	97.5	12.2 ± 0.77^**
PAB2+/−pab8	48.8 ± 1.52	99.4	38.3 ± 8.14^**	98.3	15.9 ± 0.64^*
PAB8+/−pab2	28.1 ± 4.08^**	58.1^**	54.0 ± 3.61	96.3	8.87 ± 0.74^**

Notes.

* , P < 0.05;

** , P < 0.001.

Table 3. Class II PABP members negatively regulate root growth.

	Root length at 4 d (mm)	Root length at 6 d (mm)
WT	7.47 ± 0.61	13.11 ± 0.81
pab2	11.26 ± 0.99^**	18.11 ± 1.20^**
pab4	8.89 ± 0.74^**	15.63 ± 1.01^**
pab8	8.74 ± 1.15^**	15.84 ± 1.74^**
pab2/8	9.05 ± 0.78^**	15.26 ± 0.65^**
pab4/8	8.42 ± 0.69^**	14.84 ± 0.69^**
pab2/4	7.89 ± 0.94^*	13.16 ± 0.96

Note.

* , P < 0.05;

** , P < 0.001.

Table 4. Class II PABP members are required to support expression from Luc mRNA.

	Luciferase expression (Luc/min/mg protein^a)
Mutant line	Seedlings	Relative expression	Adult leaves	Relative expression	Flowers	Relative expression
WT	260766 ± 13314	1	484726 ± 77978	1	1226031 ± 61893^**	1
pab2	82857 ±18811^**	0.318	97588 ±18355^*	0.201	170351 ± 5268^**	0.139
pab4	3928 ± 411^**	0.0151	8558 ± 655^**	0.0177	3804 ± 95.6^**	0.00310
pab8	50340 ± 5156^**	0.193	142230 ±22410^*	0.293	85775 ± 2779^**	0.0700
pab2/8	4134 ± 907^**	0.0159	6870 ± 600^**	0.0142	19839 ± 1196^**	0.0162
pab4/8	391 ± 76.6^**	0.00150	1535 ± 201^**	0.00317	3776 ± 731^**	0.00308
pab2/4	371 ± 25.0^**	0.00142	1065 ± 176^**	0.00220	5304 ± 640^**	0.00433

Notes.

^a All luciferase expression data are normalized for equal amounts of total protein.

* , P < 0.005;

** , P < 0.001.

Table 5. Class II PABP members are required to support Luc transcript level.

	Relative Luc transcript level^a
Mutant line	Seedlings	Adult leaves	Flowers
WT	1	1	1
pab2	0.688 ±0.244	0.319 ±0.0781^**	0.0795 ± 0.0317^**
pab4	0.0136 ± 0.00682^**	0.0291 ± 0.00506^**	0.000114 ± 0.0000536^**
pab8	0.161 ± 0.0853^**	0.272 ±0.0649^**	0.0423 ±0.00419^**
pab2/8	0.0491 ± 0.0246^**	0.0206 ± 0.00308^**	0.00124 ± 0.000467^**
pab4/8	0.0459 ± 0.0241^**	0.0246 ± 0.00496^**	0.00027 ± 0.000155^**
pab2/4	0.0340 ± 0.00761^**	0.0170 ± 0.00186^**	0.000083 ± 0.0000135^**

Notes.

^a As determined by qPCR for Luc mRNA in each line normalized to the PP2A reference mRNA and expressed relative to the WT level.

** , P < 0.001.

Table 6. Class II PABP members are required to support expression from Ω-Luc mRNA.

	Luciferase expression (Luc/min/mg protein^a)
Mutant line	Seedlings	Relative expression	Adult leaves	Relative expression	Flowers	Relative expression
WT	1168609 ± 164358	1	244319 ± 6257	1	1115397 ± 214567	1
pab2	1774 ± 69.2^**	0.00152	102 ±14.4^**	0.000417	337 ± 81.0^**	0.000302
pab4	1601 ± 284^**	0.00137	39.3 ± 5.03^**	0.000161	181 ± 95.8^**	0.000162
pab8	18517 ± 1157^**	0.0158	2216 ±374^**	0.00907	2911 ± 169^**	0.00261
pab2/8	9291 ± 367^**	0.00795	125 ± 24.2^**	0.000512	159 ± 31.3^**	0.000143
pab4/8	188 ± 40.4^**	0.000161	22.7 ± 13.3^**	0.0000929	46.7 ± 7.02^**	0.0000419
pab2/4	2985 ± 616^**	0.00255	34 ± 5.29^**	0.000139	207 ± 82.4^**	0.000186

Notes.

^a All luciferase expression data are normalized for equal amounts of total protein.

** , P < 0.001.

Table 7. Class II PABP members are required to support W-Luc transcript level.

	Relative W-Luc transcript level^a
Mutant line	Seedlings	Adult leaves	Flowers
WT	1	1	1
pab2	0.018073 ± 0.01^**	0.00619 ± 0.00189^**	0.00584 ± 0.000433^**
pab4	0.02141 ± 0.00905^**	0.00565 ± 0.00274^**	0.00621 ± 0.000351^**
pab8	0.03667 ± 0.0169^**	0.00212 ± 0.000733^**	0.00596 ± 0.00200^**
pab2/8	0.025197 ± 0.0128^**	0.00645 ± 0.00284^**	0.00549 ± 0.00235^**
pab4/8	0.020907 ± 0.0144^**	0.00241 ± 0.00117^**	0.00141 ± 0.000328^**
pab2/4	0.0758 ± 0.0355^**	0.00431 ± 0.00143^**	0.00236 ± 0.00110^**

Notes.

^a As determined by qPCR for Ω-Luc mRNA in each line normalized to the PP2A reference mRNA and expressed relative to the WT level.

** , P < 0.001.

Figure 6. Expression from class II PABP genes in class II PABP mutants. qPCR was performed for PAB2 (black bars), PAB4 (gray bars), and PAB8 (white bars) in 8 day old WT seedlings and class II PABP mutants. Expression for each gene is reported relative to the level in WT seedlings which is set at a value of 1.